Misidentification of Lophomonas Blattarum: Methodological Flaws and Taxonomic Confusion in Molecular Diagnostics

Abstract

Background:

Lophomonas blattarum is a protozoan, which is controversial regarding pathogenicity in the respiratory disease, and identification methods. This study investigated the presence of this protozoan in patients admitted to the intensive care unit (ICU) to assess these controversies.

Materials and Methods:

Samples of 83 patients hospitalized in the ICU ward of Afzalipoor, Shafa, and Shahid Bahonar hospitals (Kerman, Iran) were collected. The samples were examined using microscopy and real-time polymerase chain reaction (PCR) to check the presence of Lophomonas blattarum. Then, the efficiency of the method used was investigated using bioinformatics studies and the presence of Trichomonas tenax in the samples was investigated.

Results:

Of 83, 38 (46%) were female, 45 (54%) were male, and their age was 46.6 ± 14.45 years. Microscopic examination did not show any in the samples. The real-time PCR method showed 16 positive samples with primers that had been reported in other studies for Lophomonas blattarum. The bioinformatics study showed the method introduced in the other studies lacks the efficiency and specificity, and there is no information in the databases to design a molecular method based on the PCR for identification of Lophomonas blattarum. The results of examining the presence of Trichomonas tenax using the real-time PCR method showed that 16 samples with a positive result for Lofomonas blattarum contained Trichomonas tenax, which indicated that a misidentification had probably occurred.

Conclusion:

The current methods that are used to identify Lophomonas blattarum do not have the sensitivity and specificity required to identify this protozoan.

INTRODUCTION

Lophomonas blattarum (L. blattarum) belonged to the Parabasalids group of flagellated protists and was first described by S. Stein in 1860. It is an anaerobic multiflagellated protozoan that lives in the digestive tract of some arthropods, such as cockroaches, and contaminates the environment through the secretions and feces of these organisms.[1] Some studies state that the Lophomonas genus can damage the respiratory tract and thus cause diseases such as bronchitis and pneumonia, especially in people with a compromised immune system such as acquired immunodeficiency syndrome (AIDS), malignancies, users of immunosuppressing drugs and corticosteroids, and hospitalized patients.[2,3,4]

However, some studies have had opposite results, questioned the effectiveness of the methods used to identify L. blattarum in other studies, and rejected the results of other studies.[5,6] The flaws in the detection of L. blattarum in respiratory tract fluid samples are one of the reasons for this disagreement. The morphological description of this microorganism by light microscope includes a round to oval shape with a diameter of about 20 to 60 micrometers, a bunch of double flagella at the anterior end, the plasticity of the cytoplasm, containing large granules and some phagocytic vacuoles, and a nucleus that in many occasion was not visible.[7,8] On one hand, it is not easy to detect this organism in samples by light microscopy method, and on the other hand, this organism is easily confused with other microorganisms similar in shape and appearance, such as Parabasalia group members and epithelial cells in the sample. In addition, its transformation into a cyst form increases the difficulty of its diagnosis.[5,9,10]

Due to the difficulty of detecting L. blattarum by light microscopy, more accurate diagnostic techniques based on molecular identification seem necessary. Recently, a diagnostic method based on the polymerase chain reaction (PCR) technique has been provided to identify this organism by identifying a fragment of this microorganism’s small subunit ribosomal RNA sequence.[11]

In this study, two methods of light microscopy and molecular-based real-time PCR detection method were used to identify L. blattarum to check the effectiveness and biases of these two methods in identifying the L. blattarum. In addition, bioinformatics and real-time PCR studies were used to verify the authenticity of the PCR method used in other studies.

MATERIALS AND METHODS

This study was approved by the Ethics Committee of Kerman University of Medical Sciences and in line with the guidelines of the Declaration of Helsinki.[12] The study workflow is summarized in Figure 1 to make it easier to understand. The patient’s companions were explained about the study and its objectives, and a written consent form was obtained from them. All samples were collected from intensive care unit (ICU) patients hospitalized in Afzalipour, Shafa, and Shahid Bahonar hospitals in the city of Kerman from 2019 to 2021, and since this study was carried out during the outbreak of Coronavirus disease 2019 (COVID-19), only patients with SARSCoV2 PCR test negative were selected. Mini-bronchoalveolar lavage (Mini-BAL) samples were collected from 83 patients.

Figure 1.

Summary of the workflow done in this study

One of the goals of this study was to check the efficiency of L. blattarum identification methods. One of the biggest concerns about identifying L. blattarum is reporting false cases. Therefore, the experts of the parasitology department were not informed about the study to examine the samples blindly and prevent biases in the study. A wet slide was prepared from the samples and was inspected by a light microscope. The remaining samples were kept at a temperature of -70°C until the time of molecular assay.

For L. blattarum detection by real-time PCR, DNA was extracted from the frozen sample using the Tissue DNA genomic DNA extraction mini kit (YATA, Iran). The quality of the extracted DNA was confirmed by the Epoch reader using a Take3 Micro-volume Plate and dedicated software for analysis of 260/280 ratio (Biotek, USA). The real-time PCR assay was performed using the reported primers[11] (Forward primer: 5’-GAGAAGGCGCCTGAGAGAT-3’, Reverse primer: 5’-ATGGGAGCAAACTCGCAGA-3’) and by using the SYBR Green qPCR master mix (YATA, Iran). The tests were performed with a total volume of 20 ul, including 10 ul of master mix, 1 ul of 10 uM primers each, 1 ul of the DNA sample, and 7 ul of water using the Rotor-Gene 6000 cycler instrument (Corbett, Australia). Test conditions include a 3-minute step at 95°C, followed by 40 cycles of 3 steps, including melting at 95°C, annealing at 60°C, and extension at72°C for 20 seconds each, and data acquisition on the green channel at the end of extension step. The samples were subjected to the melting curve analysis incrementally from 65°C to 95°C with one °C steps to assess the identity and purity of amplified products.

The sequence of the used primers was checked by the National Center for Biotechnology Information (NCBI) Primer-Basic Local Alignment Search Tool (Primer-BLAST) web-based software to assess the specificity of the real-time PCR primers. The “Organism” part was left blank to maximize the results, and the “nr” database was selected. Other parameters were used unchanged.

To obtain the recorded sequences of L. blattarum, the keyword “Lophomonas blattarum” was searched in the NCBI taxonomy browser. All obtained sequences were subjected to an alignment test using the NCBI nucleotide-BLAST algorithm, and their similarity level was determined. After that, a part of the genetic sequence that was the most common in the obtained sequences was analyzed by the nucleotide-NCBI algorithm to find the degree of similarity between the recorded sequences for L. blattarum and other sequences in the NCBI database. The nucleotide-BLAST was optimized for highly similar sequences (megablast) and used without changing the default parameters, except when pairwise comparing recorded sequences, where the “Align two or more sequences” option was selected. Also, the sequence with the longest length was chosen as “Query” and other sequences as “Subject”.

Since the sample used in this study was collected from the respiratory tract, we decided to study the presence of Trichomonas tenax (T. tenax), a parasite with high genetic similarity usually found in the oral cavity and sometimes in the respiratory tract, using the real-time PCR method. The primers were designed using Primer-BLAST web software (Forward primer: 5’- CCTTGGGTCCTTCAGGATAATTC-3’, Reverse primer: 5’-ACGCTAGACAGGTCAACCCA-3’). The primers were designed to amplify a 73 bp amplicon and for the small subunit ribosomal RNA gene of T. tenax with accession number MZ388570.1 and aligned against similar sequences. The real-time PCR used for T. tenax was similar to the method used for L. blattarum, with the difference that the desired amplicon length was 73 and a dedicated primer explicitly designed for T. tenax was used.

RESULTS

Of 83 patients examined in this study, 38 (46%) were female, and 45 (64%) were male. The average age of the participants was 46.5 ± 14.45 years. In the light microscope examination, the laboratory experts who were blind to the study observed no parasite or suspicious or similar organism. The results obtained from the real-time PCR method to detect L. blattarum in the DNA sample extracted from the Mini-BAL sample showed 16 positive samples. The melting curve of the samples showed a single peak, which indicated the identity and purity of amplified products.

Primer-BLAST software aligning results showed that primers that were supposed to detect L. blattarum were not specific. The primers used are designed for the identification and amplification of a part of the small subunit ribosomal RNA gene, which is the same in many species in the phylum of “parabasalids” including Trichomonas, Tritrichomonas, Runanympha, Macrotrichomonas, Devescovina, Honigbergiellida, Hoplonympha, Tetratrichomonas, Parabasalia, Simplicimonas, Trichomitopsis, Metadevescovina, Macrotrichomonoides, Kofoidia, Monocercomonas, Simplicimonas, Calcaritermes, Cryptotermes, Heterotermes, Cthylla, Coronympha, and Pentatrichomonas. The complete results of the Nucleotide-BLAST alignment are in the supplementary file.

Sixteen nucleotide sequences for L. blattarum were recorded in NCBI, all of which were small subunit ribosomal RNA genes. For alignment, the sequence with the longest length (JX020505.1) was selected as “Query” and other sequences as “Subject”. The sequence ID (Accession Number), length, query coverage percent, identity percent, numbers and positions of mismatch nucleotide, and the country where the sequence was reported are given in Table 1. There was a 4 to 6 nucleotide difference between the Query and subjects. Multiple Sequence Alignment viewer (MSA viewer) results are shown in Figure 2. The full results are in the file.

Table 1.

The results were obtained by aligning the sequences registered for L. blattarum in the NCBI database

Sequence ID		Length in base pair		Query coverage percent		Identity percent		Number of different nucleotides (positions)		Country
Query
JX020505.1		328		-		-		-		Thailand
Subjects
MZ093069.1		203		61		97.54		5(182,195,209,224,273)		Iran
MZ093070.1		202		61		97.52		5(182,195,209,224,273)		Iran
MZ093074.1		201		61		97.51		5(182,195,209,224,273)		Iran
MZ093077.1		201		61		97.51		5(182,195,209,224,273)		Iran
MZ093075.1		200		60		97.50		5(182,195,209,224,273)		Iran
MZ093078.1		200		60		97.50		5(182,195,209,224,273)		Iran
MZ093071.1		199		60		97.49		5(182,195,209,224,273)		Iran
MZ093072.1		199		60		97.49		5(182,195,209,224,273)		Iran
MZ093073.1		196		59		97.45		5(182,195,209,224,273)		Iran
MZ093076.1		195		59		97.45		5(182,195,209,224,273)		Iran
OL477431.1		193		58		96.86		6(151,182,195,209,224,273)		Iran
OL477422.1		186		56		97.31		5(182,195,209,224,273)		Iran
OL477423.1		185		56		96.74		6(182,195,198,209,224,273)		Iran
OL477421.1		177		53		97.18		5(182,195,209,224,273)		Iran
MZ093079.1		139		42		97.10		4(151,182,195,209)		Iran

Figure 2.

The results from checking the sequences registered for L. blattarum in the NCBI database were obtained using the Multiple Sequence Alignment Viewer (MSA viewer) services. The image is limited to the part where there are sequence differences. Small red squares indicate different positions and types of nucleotides

Based on the mismatches, 16 sequences were divided into five categories so that the sequences in each category were the same and their sequences were aligned.

The results of the real-time PCR test for the presence of T. tenax showed that 16 samples that tested positive for L. blattarum were also positive for T. tenax. The melting curve of the samples showed a single peak, which indicated the identity and purity of amplified products.

DISCUSSION

In this study, the identification of L. blattarum was investigated using light microscopic and molecular real-time PCR methods using the information provided in previous studies and the accuracy and efficiency of the data used. In addition, the presence of T. tenax parasites in Mini-BAL samples was evaluated.

Considering that the microscopic examiners were blind to the study’s objectives, the presence of L. blattarum via the microscopic examination was not confirmed. In most studies, the detection and confirmation of L. blattarum have been done using light microscopy.[5,13,14] While some researchers reported the light microscope method as the simple and gold standard for diagnosing L. blattarum,[15,16] in a controversial study using the electron microscopy technique, Ran Li and his colleague announced the high possibility of reporting false positive cases. They stated that all reported infections were diagnosed only by light microscopic morphology rather than electron microscopy, isolation, culture, or molecular methods. They mentioned that the images presented in these articles were all consistent with the microscopic features of bronchial ciliated epithelial cells. In a controversial statement, they stated that bronchopulmonary infection with L. blattarum was probably misdiagnosed.[9]

Due to its nature, light microscopic examination can be subjective, error-prone, and controversial, and the perhaps existence of such reasons encouraged researchers to seek and use more accurate molecular methods, such as PCR, to diagnose L. blattarum. For the first time, in 2019, Fakhar. M and his colleagues reported a PCR molecular method to identify L. blattarum in a bold claim and an article entitled “First Molecular Diagnosis of Lophomoniasis: the End of a Controversial Story”. Four articles have been published using the PCR method suggested by Fakhar. M.[11]

Therefore, we decided to check the presence of L. blattarum in the samples using the information from the reported PCR method. The results confirmed the presence of parasites in 16 (19%) samples. However, to ensure the quality of our study, we decided to examine the sequence of the primers used. The results were alarming and unexpected (details in the material and method section), making the real-time PCR method and the result unreliable. The reported primers and the PCR method designed for L. blattarum were not specific. Obtaining such results led us to examine the sequences recorded for L. blattarum in the databases, which also showed that the sequences recorded for L. blattarum are very similar to a large number of similar parasites (details in Material and Method section), so it was concluded that the design of primers is not possible to identify L. blattarum precisely.

We had samples that were found to be devoid of parasitic organisms by light microscopy, but some of them contained the genetic content of the small subunit ribosomal RNA gene, common among many species of the “parabasalids” phylum. Testing the presence of T. tenax in the samples by real-time PCR showed that the samples that tested positive for L. blattarum by real-time PCR were also positive for T. tenax. Although this result most likely indicates that the positive samples of L. blattarum are T. tenax, we cannot reject the existence of L. blattarum in the same way that it is impossible to deny the existence of any other microorganisms.

The results of this study and the review of previous studies emphasize several concerns. Due to the high background nature of the respiratory track sample and the possibility of error, the light microscopy method has neither sensitivity nor specificity to identify the L. blattarum. The situation of the PCR molecular method presented based on erroneous information creates a much more serious situation. Errors seem to have their roots in the wrong recognition, interpretation, and use of molecular information and their conversion into molecular methods. The molecular method presented based on the PCR technique has no specificity for the identification of the parasite L. blattarum. Currently, no sequence in the NCBI database can be used to design a specific PCR method, and the providers of the PCR method could perhaps have avoided making a mistake and presenting a wrong method by performing a simple alignment in genetic databases.

CONCLUSION

While the number of manuscripts related to L. blattarum is increasing, there are considerable concerns regarding the identification methods used. Based on current data and contradictions regarding the identification of L. blattarum, the arguments about the association of this parasite with the proposed respiratory diseases are unclear. Even if a reliable method is introduced to identify this parasite and prove its association with some respiratory diseases, establishing pathogenicity and confirming its cause-and-effect relationship requires extensive and targeted studies. If needed, it is suggested that after isolating the parasite from the primary host and culturing the parasite, the genetic content of L. blattarum should be sequenced, and a specific PCR test should be designed for specific identification.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Kerman University of Medical Sciences with the code IR.KMU.AH.REC.1399.43

Conflicts of interest

There are no conflicts of interest.

Funding Statement

This work was supported by the Kerman University of Medical Sciences.