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  • Research article
  • Open Access
  • Open Peer Review

Use of nested PCR for the detection of trichomonads in bronchoalveolar lavage fluid

BMC Infectious Diseases201919:512

https://doi.org/10.1186/s12879-019-4118-9

  • Received: 16 March 2019
  • Accepted: 21 May 2019
  • Published:
Open Peer Review reports

Abstract

Background

The methods routinely used to detect trichomonads in the lungs are not sensitive enough, and an effective method is urgently needed.

Method

Primers were first designed to match the conserved area of the 18S rRNA gene of trichomonads. Then, nested PCR was carried out to detect trichomonads in bronchoalveolar lavage fluid (BALF). Finally, all positive specimens were subjected to DNA sequencing and phylogenetic analysis.

Results

Among 115 bronchoalveolar lavage fluid samples, ten samples tested positive in nested PCR (10/115), while no samples were positive in wet mount microscopy (0/115) (P < 0.01). Among the ten positive specimens, two were identified as Tetratrichomonas spp. and the other eight as Trichomonas tenax in phylogenetic analysis.

Conclusions

Nested PCR is an effective way to detect trichomonads in bronchoalveolar lavage fluid.

Keywords

  • Bronchoalveolar lavage fluid
  • Nested PCR
  • Trichomonad
  • Lung

Background

Trichomonads are a type of parasitic flagellate protozoan of the genus that are found in the digestive and reproductive systems of man and animals. They frequently colonize the human lungs, but this condition is unfamiliar to most physicians [1, 2]. Several kinds of trichomonads can infect the lungs, such as Trichomonas vaginalis, Trichomonas tenax, Pentatrichomonas hominis, and Tetratrichomonas spp [36]. At present, microscopic detection is the most common approach for testing trichomonads in the clinic. However, numerous factors can make their recognition very difficult, if not impossible. [7, 8] First, immobility of trichomonads due to low temperature or for long periods in vitro can reduce the sensitivity. Second, many epithelial cells of lung alveoli move similarly to trichomonads, which could impede trichomonad detection [1, 9]. Third, trichomonads can develop into an amoeboid form, making them unrecognizable [10]. Therefore, there is an urgent need to establish a sensitive molecular method to diagnose pulmonary trichomoniasis. However, doing so is further complicated by the fact that numerous trichomonad species can infect human lungs. Existing gene based methods have been used to detect single types of trichomonads [4, 1113]. However, not all species can be tested at a single time. To overcome this issue, all 18S rRNA gene sequences of different kinds of trichomonads were downloaded from NCBI, and then the most conserved area of this gene was identified through sequence alignment. Primers were then designed to cover this conserved region. To determine the prevalence of Trichomonas infection in the lungs, bronchoalveolar lavage fluids (BALF) from 115 cases were tested using nested PCR and microscopy. Furthermore, phylogenetic analysis was performed to determine which type of trichomonad is most likely to infect human lungs.

Methods

Bronchoalveolar lavage specimen collection

One hundred fifteen BALF samples were obtained from 113 patients, who visiting pulmonary specialists, at the First Affiliated Hospital of Wenzhou Medical University between 2017 and 2018 and were analyzed by investigators blinded to the clinical data. These BALF samples were obtained by fibre-optic bronchoscopy. When using bronchoscopy for alveolar lavage, oral contact is avoided as much as possible. Each patient was given 20 to 120 ml of normal saline irrigation based on the situation. Then, each sample was collected in 400 μl of sediment after centrifugation at 400 g for 5 min, after which the supernatant was discarded.

Microscopy

Microscopy was utilized to detect trichomonads. A wet smear was created with 50 μl of sediment, followed by direct observation under a microscope. This direct microscopy was performed within two hours of BALF collection. Quality control of microscopy throughout the study was maintained by experienced microscopists who had more than 10 years’ work experience. All slides were rechecked within 15 min by experienced microscopists. Microscopy was performed at a magnification of 400×, and 20 fields were examined. The flagellated protozoa, which are as large as one to four white blood cells, were considered trichomonads. A negative diagnosis was made when no trichomonads were found.

Genomic DNA extraction and polymerase chain reaction

Genomic DNA was extracted using a TIANamp Blood DNA Kit (Tiangen, China) from 200 μl of BALF sediment. The 18S rRNA gene was first amplified using the primers TRC1-F (5′-GGTAATTCCAGCTCTGCG-3′) and TRC1-R (5′-TGGTAAGTTTCCCCGTGT-3′). PCR was performed in a volume of 20.0 μl with 1.0 μl of DNA template, 0.3 μM of each primer, 2.0 μl of 10× PCR buffer, 2.0 units of DNA polymerase, and 0.2 mM dNTP mix. The reaction conditions consisted of initial denaturation at 98 °C for 2 min; 20 cycles of 98 °C for 10 s, 53 °C for 30 s and 68 °C for 30 s; and a final extension step for 5 min at 68 °C. Subsequently, the 18S rRNA gene was amplified by the primers TRC2-F (5′-GTTAAAACGCCCGTAGTC − 3′) and TRC2-R (5′-CCAGAGCCCAAGAACTAT-3′). PCR was performed in 20.0 μl with 0.4 μl of DNA template derived from the product amplified by TR1-F and TR1-R, 0.3 μM of each primer, 2.0 μl of 10× PCR buffer, 2.0 units of DNA polymerase, and 0.2 mM dNTP mix. The reaction conditions consisted of initial denaturation at 98 °C for 2 min; 35 cycles of 98 °C for 10 s, 54 °C for 30 s and 68 °C for 30 s; and a final extension step for 5 min at 68 °C.

Sensitivity and specificity of the nested PCR assay

To compare the detection limit of the nested PCR assay with microscopy, serially diluted BALF containing T. vaginalis (0.1, 1, 10, 102 trichomonads/μl) were used as the target, and all samples were analyzed in triplicate. The specificity of the nested PCR was assessed by testing the DNA samples from protozoa, including T. vaginalis, T. tenax, T. hominis, Giardia intestinalis (G. lamblia), Toxoplasma gondii and human DNA.

Molecular phylogenetic analysis

Through separation by agarose gel electrophoresis, a single amplicon of approximately 400 bp was obtained. After that, the amplicon was sequenced in both directions using the sequencing primers TRC2-F and TRC2-R. Finally, the sequences were used as queries for BLAST searches in the GenBank database (http://www.ncbi.nlm.nih.gov/Blast.cgi) to identify the types of trichomonads.

To further identify the species, the sequences were compared with 80 trichomonad 18S rRNA, which were acquired from the National Center for Biotechnology Information database. Multiple alignments were performed using the Clustal W program. The evolutionary history was inferred using the neighbor-joining method based on the Tamura-Nei model. [2, 14] The percentage of replicate trees in which the associated taxa clustered together was computed with a bootstrap test (1000 replicates) [15]. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method [2] and are expressed in units of the number of base substitutions per site. The analysis involved 91 nucleotide sequences. The codon positions included were 1st + 2nd + 3rd + noncoding. All positions containing gaps and missing data were eliminated. There were a total of 314 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [16].

Statistical analysis

One hundred fifteen samples were subjected to nested PCR and microscopic testing. The data were analyzed using Microsoft Excel and IBM SPSS 20.0. Fisher’s exact test was used to compare frequency data, and the Clopper-Pearson exact method based on the beta distribution was used to determine the 95% confidence intervals for proportions. Differences were considered statistically significant when P < 0.05.

Result

Detection results for different kinds of trichomonads

To test the capabilities of the methods to detect trichomonads, the three most common trichomonads and two other common parasites in humans were tested. In gel electrophoresis after nested PCR, T. vaginalis, T. tenax and T. hominis displayed distinctive bands, while G. lamblia,T. gondii and the negative control, human DNA, displayed no band, and the positive control, a sample of T. vaginalis, displayed a distinctive band. (Fig. 1).
Fig. 1
Fig. 1

Detection of different trichomonads by nested PCR: GoldView-stained agarose gel showing the test results of trichomonad protozoa and other parasites. M, 100-bp DNA molecular size markers; +, positive control (Trichomonas vaginalis); −, negative control (human DNA); 1, Toxoplasma gondii; 2, Giardia intestinalis; 3, Pentatrichomonas hominis; 4, Trichomonas vaginalis; 5, Trichomonas tenax

Sensitivity of nested PCR

The sensitivity analysis showed that the detection limit of the nested PCR assay was 100 trichomonads/mL of BALF (Fig. 2a). For microscopy, the detection limit was 10,000 trichomonads/mL of BALF (Fig. 2b).
Fig. 2
Fig. 2

Assessment of the sensitivity of nested PCR for T. vaginalis in comparison with the microscopy test. a Sensitivity analysis of PCR assay on agarose gel. b Sensitivity analysis of microscopy. M, 100-bp DNA molecular size markers; +, positive control (T. vaginalis); −, negative control (human DNA). 1–4, Serial diluted BALF containing T. vaginalis (0.1, 1, 10, 102 trichomonads/μl); the red square indicates positive results for microscopy; the green square indicates negative results for microscopy

Test results for bronchoalveolar lavage fluid

One hundred fifteen BALF samples were obtained from 113 consecutive patients who visited pulmonary specialists where investigators were blinded to the clinical data. The majority of patients were men (60%). The median age was 56 years (interquartile range, 47 to 66). Upon nested PCR analysis, 10 of the 115 specimens gave positive results. In addition, the negative control, human DNA, gave no band, while the positive control, T. vaginalis, had a distinctive band. (Fig. 3). None of the patients who tested positive were found to have a history of HIV infection or radiation treatment by retrospective analysis.
Fig. 3
Fig. 3

Gel image of positive samples: GoldView-stained agarose gel showing the PCR amplicons of the 18S RNA gene of trichomonad species; M, 100-bp DNA molecular size markers; +, positive control (T. vaginalis); −, negative control (human DNA); 1–10, positive samples

Sequencing results

All PCR products of the ten positive samples were sequenced by a commercial organization using the TCR2-F and TCR2-R primers. The sequences were then used as queries for BLAST searches in the GenBank database (http://www.ncbi.nlm.nih.gov/Blast.cgi), and all sequences had no less than 99% homology (~ 380 bp) with Trichomonas sequences.

Difference in positivity rates between the two methods

One hundred fifteen consecutive BALF samples obtained from 113 patients were tested by nested PCR and microscopy. Ten samples were positive in nested PCR, while none of the samples were positive in the microscopic test. (P < 0.01) (Table 1).
Table 1

Differences in positive rates between nested PCR and microscopy

 

Positive (n)

Negative (n)

P

Nested PCR

10

105

 

Microscopy

0

115

< 0.01

Molecular phylogenetic analysis

The sequences of the ten positive samples were compared with 80 trichomonad 18S RNA sequences acquired from the NCBI and a positive control. The analysis involved 91 nucleotide sequences. The codon positions included were 1st + 2nd + 3rd + noncoding. All positions containing gaps and missing data were eliminated. There were a total of 314 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [16]. As shown in Fig. 4, two samples were classified as Tetratrichomonas, and the others were classified as Trichomonas tenax. (Fig. 4).
Fig. 4
Fig. 4

Evolutionary relationships of the trichomonads. Phylogenetic tree of several members of the family Trichomonadidae based on 18S RNA sequences. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The sequences of the ten positive specimens, shown by the red dots in the figure, were compared with 80 trichomonad sequences available in the National Center for Biotechnology Information database. The GenBank accession numbers are shown in parentheses. T. vaginalis, shown by a pink rhomboid in the figure, was used as a positive control and was grouped in the appropriate species clades

Discussion

In most respiratory tract infections, the causative pathogen remains unknown, so there is an urgent need to improve detection methods. Trichomonads are a type of protozoan with several flagella, and they are microscopic in size. [1719]. Thus, trichomonads cannot be directly observed by the naked eye. Microscopy is still routinely used to detect trichomonads. [19]. However, it is sometimes difficult to distinguish trichomonads for several reasons. First, some normal ciliated columnar epithelial cells can move like protozoa in wet films. Second, the BALF collected from patients infected by trichomonads always contains many inflammatory cells, which will interfere with the detection of trichomonads. Third, trichomonads may lose mobility if not detected in a timely manner. Therefore, it is sometimes difficult to accurately detect trichomonads with routine methods in the clinic.

Our studies have shown that the nested PCR that we designed can detect trichomonads in BALF specimens in a sensitive and specific manner. Among the 115 BALF samples analyzed, ten samples were positive in nested PCR (10/115), while none of the samples were positive in a wet microscopy test (0/115) (P < 0.01). (Table 1) The positivity rate of nested PCR was significantly higher than that of microscopy. Moreover, nested PCR was very specific. Upon BLAST searches in the GenBank database (http://www.ncbi.nlm.nih.gov/Blast.cgi), all of the sequences revealed no less than 99% homology (~ 380 bps) with Trichomonas sequences.

Moreover, our study suggests that an unidentified Tetratrichomonas sp. and T. tenax are very important species among the trichomonads that can infect humans. Similar to previous studies, we also showed that Trichomonas tenax, a common inhabitant of the human oral cavity, is the most common species to infect human lungs [3, 20]. Tetratrichomonas spp. are usually reported as pathogenic agents in animals rather than in humans [21, 22]. In our study, however, two of the ten positive samples were identified as Tetratrichomonas spp. Whether these Tetratrichomonas spp. can spread between humans and animals need further confirmation. Further studies should seek to elucidate the mechanisms of the transmission and pathogenesis of these Tetratrichomonas spp.

One limitation of our study is that we cannot be sure that this nested PCR method can detect trichomonads for which no 18S RNA sequences exist in GenBank.

Conclusion

In conclusion, the results of our study demonstrate that nested PCR is an effective way to detect trichomonads in BALF. Moreover, our study suggests that Tetratrichomonas as well as T. tenax is a very important species among the trichomonads that can infect humans. Our findings support the application of nested PCR in the detection of trichomonas in the clinic. Further research in this area should seek to elucidate the mechanisms of the transmission and pathogenesis of Tetratrichomonas spp.

Abbreviations

BALF: 

Bronchoalveolar lavage fluid

NCBI: 

National Center for Biotechnology Information database

PCR: 

polymerase chain reaction

Declarations

Acknowledgements

We thank the patients for their participation and cooperation and the staff members of the clinical laboratory of the First Affiliated Hospital of Wenzhou Medical University for their help in the collection of samples.

Funding

This work was made possible with aid from the First Affiliated Hospital of Wenzhou Medical University. The funding agencies had no involvement in study design, data collection, data analysis, and data interpretation.

Authors’ contributions

DH. and TZ. contributed to the study design. CL., FY. and YL. collected the samples and clinical data. CL., YL. and XL. performed the microscopic tests. CL. and YL. performed the genomic DNA extraction and polymerase chain reaction. XX. conducted the molecular phylogenetic analysis. D.H. and TZ. analyzed the data and wrote the manuscript. CL., FY. and YL. contributed equally to this work. All authors read and approved the final manuscript.

Ethics approval and consent to participate

All procedures performed in studies involving humans were in accordance with the ethical standards of the institution or practice in which the studies were conducted. This article does not contain any studies with animals performed by any of the authors. The study was also approved (approval number: 2018058) by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University. Written informed consent was obtained from all individual participants included in the study.

Consent for publication

Not applicable.

Competing interests

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, Zhejiang Province, China
(2)
Wenzhou Medical University, Wenzhou, 325000, Zhejiang Province, China

References

  1. Duboucher C, Caby S, Chabe M, Gantois N, Delgado-Viscogliosi P, Pierce R, Capron M, Dei-Cas E. Viscogliosi E: [human pulmonary trichomonoses]. Presse Med. 2007;36(5 Pt 2):835–9.View ArticleGoogle Scholar
  2. Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci U S A. 2004;101(30):11030–5.View ArticleGoogle Scholar
  3. Chiche L, Donati S, Corno G, Benoit S, Granier I, Chouraki M, Arnal JM, Durand-Gasselin J. Trichomonas tenax in pulmonary and pleural diseases. Presse Med. 2005;34(19 Pt 1):1371–2.View ArticleGoogle Scholar
  4. Lopez-Escamilla E, Sanchez-Aguillon F, Alatorre-Fernandez CP, Aguilar-Zapata D, Arroyo-Escalante S, Arellano T, Moncada-Barron D, Romero-Valdovinos M, Martinez-Hernandez F, Rodriguez-Zulueta P, et al. New tetratrichomonas species in two patients with pleural empyema. J Clin Microbiol. 2013;51(9):3143–6.View ArticleGoogle Scholar
  5. Salvador-Membreve DM, Jacinto SD, Rivera WL. Trichomonas vaginalis induces cytopathic effect on human lung alveolar basal carcinoma epithelial cell line A549. Exp Parasitol. 2014;147:33–40.View ArticleGoogle Scholar
  6. Hersh SM. Pulmonary trichomoniasis and trichomonas tenax. J Med Microbiol. 1985;20(1):1–10.View ArticleGoogle Scholar
  7. Kamaruddin M, Tokoro M, Rahman MM, Arayama S, Hidayati AP, Syafruddin D, Asih PB, Yoshikawa H, Kawahara E. Molecular characterization of various trichomonad species isolated from humans and related mammals in Indonesia. Korean J Parasitol. 2014;52(5):471–8.View ArticleGoogle Scholar
  8. Dos Santos CS, de Jesus VLT, McIntosh D, Carreiro CC, Batista LCO, do Bomfim Lopes B, Neves DM, Lopes CWG. Morphological, ultrastructural, and molecular characterization of intestinal tetratrichomonads isolated from non-human primates in southeastern Brazil. Parasitol Res. 2017;116(9):2479–88.View ArticleGoogle Scholar
  9. Li R, Gao ZC. Lophomonas blattarum infection or just the movement of ciliated epithelial cells? Chin Med J. 2016;129(6):739–42.View ArticleGoogle Scholar
  10. Duboucher C, Caby S, Pierce RJ, Capron M, Dei-Cas E, Viscogliosi E. Trichomonads as superinfecting agents in Pneumocystis pneumonia and acute respiratory distress syndrome. J Eukaryot Microbiol. 2006;53(Suppl 1):S95–7.View ArticleGoogle Scholar
  11. Mahmoud MS, Rahman GA. Pulmonary trichomoniasis: improved diagnosis by using polymerase chain reaction targeting trichomonas tenax 18S rRNA gene in sputum specimens. J Egypt Soc Parasitol. 2004;34(1):197–211.PubMedGoogle Scholar
  12. Mallat H, Podglajen I, Lavarde V, Mainardi JL, Frappier J, Cornet M. Molecular characterization of trichomonas tenax causing pulmonary infection. J Clin Microbiol. 2004;42(8):3886–7.View ArticleGoogle Scholar
  13. Yao C, Ketzis JK. Aberrant and accidental trichomonad flagellate infections: rare or underdiagnosed? Trans R Soc Trop Med Hyg. 2018;112(2):64–72.View ArticleGoogle Scholar
  14. Tamura K, Nei M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol. 1993;10(3):512–26.PubMedGoogle Scholar
  15. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985;39(4):783–91.View ArticleGoogle Scholar
  16. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870–4.View ArticleGoogle Scholar
  17. Lewis KL, Doherty DE, Ribes J, Seabolt JP, Bensadoun ES. Empyema caused by trichomonas. Chest. 2003;123(1):291–2.View ArticleGoogle Scholar
  18. Carter JE, Whithaus KC. Neonatal respiratory tract involvement by trichomonas vaginalis: a case report and review of the literature. Am J Trop Med Hyg. 2008;78(1):17–9.View ArticleGoogle Scholar
  19. van Woerden HC, Martinez-Giron R. Lophomonas blattarum: is it only its morphology that prevents its recognition? Chin Med J. 2017;130(1):117.View ArticleGoogle Scholar
  20. Porcheret H, Maisonneuve L, Esteve V, Jagot JL, Le Pennec MP. Pleural trichomoniasis due to trichomonas tenax. Rev Mal Respir. 2002;19(1):97–9.PubMedGoogle Scholar
  21. Smejkalova P, Petrzelkova KJ, Pomajbikova K, Modry D, Cepicka I. Extensive diversity of intestinal trichomonads of non-human primates. Parasitology. 2012;139(1):92–102.View ArticleGoogle Scholar
  22. Xu J, Qu C, Tao J. Loop-mediated isothermal amplification assay for detection of Histomonas meleagridis infection in chickens targeting the 18S rRNA sequences. Avian Pathol. 2014;43(1):62–7.View ArticleGoogle Scholar

Copyright

© The Author(s). 2019

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