Use of the MLST technique to describe the allelic profile of Treponema pallidum in CSF samples: a systematic review

doi:10.21203/rs.3.rs-1534240/v1

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Use of the MLST technique to describe the allelic profile of Treponema pallidum in CSF samples: a systematic review

https://doi.org/10.21203/rs.3.rs-1534240/v1

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Syphilis is a sexually transmitted infection that has recently become a serious public health problem in Brazil. At the same time, the World Health Organization estimates that among 56 million individuals are infected with syphilis. Multilocus Sequence Typing consists of a high resolution molecular typing technique that is based on the amplification and sequencing of fragments of conserved genes called housekeeping (loci). The technique was recently developed and used for the study of syphilis, but there are few studies related to neurosyphilis (NS), a nosological entity with greater severity and morbidity. For Tp typing, three genes are identified: TP0136, TP0548 and TP0705. This systematic review aimed to analyze original studies that addressed the MLST technique, describing the allelic profile and discrimination of Tp clades present in each population and its relationship with clinical-epidemiological aspects, especially in neurosyphilis, through a bibliographic survey in databases with the following DECS: MLST, Multilocus Sequence Typing and Treponema pallidum. As a result, 57 articles were found, of which only 7 were selected for full reading. After reading the selected articles, only two reported CSF analysis. It is concluded that the determination of the allelic profile through MLST is a high resolution genotyping tool that allows the distinction of subspecies and species of Treponema pallidum, and can be used for differentiation of strains within each phylogenetic tree of these microorganisms. The mutation most associated with macrolide resistance is A2058G, which is commonly found in Clade SS14-like samples, in addition, the most studied gene to assess macrolide resistance is 23s rRNA. The MLST technique presents a great perspective for the deepening of questions related to the study of different strains of Tp, especially the characterization of neurotropic strains that cause NS, antibiotic resistance, phylogeny and geographic distribution.

MLST

Multilocus Sequence Typing and Treponema pallidum

Syphilis is a sexually transmitted infection that has recently become a serious public health problem in Brazil (PCDT). The World Health Organization (WHO) estimates that among 18 million organizations are infected with syphilis (1,2). Treponema pallidum subsp. pallidum (Tp) the causative agent of syphilis was first identified by Schaudinn and Hoffman in 1905 ( 3, 4 ), since then it has spread and is diagnosed globally.

The disease manifests itself in three different stages: a) primary syphilis that progresses with the appearance of painless sores on genital organs with a hardened base (hard chancre) in addition to lymphadenomegaly, which disappear spontaneously and do not leave scars, b) secondary: in this reddish spots appear on the skin, mouth mucosa, palms and soles of the feet; may be accompanied by fever; headache; malaise; inappetence; generalized lymphadenopathy, these manifestations may regress without treatment, although the disease remains active, resulting in tertiary syphilis c) tertiary: there is a long asymptomatic or oligosymptomatic period, in which the bacterium remains latent, symptoms may appear after years and with aggressiveness resulting in complications eye diseases and blindness, skin disease, osteopathies, cardiovascular manifestations and central nervous system disease leading to important neurological and mental disorders.

Another major public health problem is congenital syphilis resulting in fetal malformation, miscarriage and fetal death. Pneumonia, body wounds, osteo-articular disorders, blindness and mental development disorders appear in the first months of life.

Since 1905, several techniques have been developed and used for the diagnosis and study of Tp, however, more recently the characterization of the genetic profile has been used with relative frequency. The determination of the Tp allelic profile, through the Multilocus Sequence Typing (MLST) technique, allows the distinction of its subspecies and species, and can be used to differentiate strains within each of these phylogenetic branches of microorganisms (5).

Multilocus Sequence Typing consists of a high resolution molecular typing technique. It is based on the amplification and sequencing of conserved gene fragments called housekeeping (loci). In the case of Tp, three genes are identified: TP0136, TP0548 and TP0705. When combined, these genes correspond to about 30% of its complete genome (5). Each isolate is identified according to the allelic profile generated from the sequences of each analyzed gene. This allele profile is called Sequence Type (ST) which corresponds to a randomly generated numerical sequence (Maiden et al., 2006).

MLST is an excellent tool to assess diverse information about syphilis in clinical and epidemiological aspects (8), antibiotic resistance (9,10), genetic profiling, phylogenetics and evolutionary selection in different geographic areas (11).

MLST has been used in hundreds of clinical samples - from patients diagnosed with syphilis - in several clinical and epidemiological studies around the world, such as: Czech Republic, Switzerland, France, Cuba, China and Spain; in these countries, it was found that the presence of the A2058G mutation in the 23S rDNA gene, found in samples of the SS14 clade, is commonly associated with macrolide resistance, making the treatment of this disease difficult (5, 8-9, 12-14).

The MLST technique was recently developed and used for the study of syphilis, but there are few studies related to neurosyphilis (NS), a nosological entity with greater severity and morbidity. Sahi and his collaborators (2021) through the MLST technique found and described subtypes with greater neurotropic potential. Thus, MLST may present an enormous potential for studying this condition. NS occurs by the invasion of Tp in the Central Nervous System (CNS), in any period of syphilis, and may or may not cause manifestation of neurological symptoms. The laboratory diagnosis of NS can be performed by the positivity of the Veneral Disease Research Laboratory in the cerebrospinal fluid (VDRL-CSF), or, in its absence, by the combination of treponemal tests in the cerebrospinal fluid (CSF), in addition to the increase in cellularity and levels of CSF proteins (15).

One of the advantages of using the MLST technique is that it allows to distinguish the allelic profile based on several nucleotide differences, which allows the distinction of Tp from other treponemal species and subspecies (16). However, it is an expensive technique, requiring a laboratory structure, specialized technical training in addition to high-cost inputs and the need for constant updating of professionals.

This systematic review aimed to analyze original studies that addressed the MLST technique, describing the allelic profile and discrimination of Tp clades present in each population and its relationship with clinical-epidemiological aspects, especially in neurosyphilis.

For this survey, an electronic search was carried out in the following databases: EMBASE, Scielo, MEDLINE/PUBMED, LILACS, Cochrane Library with the descriptors in English, Portuguese and Spanish: “Treponema pallidum” and “MLST” or “Multilocus Sequence Typing” . The same descriptors were also searched in Trials databases (Clinical Trials and ICTRP) as well as in the Brazilian bases of "gray literature" in the Theses and Dissertations Catalog (CAPES) and in the Brazilian Digital Library of Theses and Dissertations. The search was limited to the period from January 2018 to January 2022, in order to select mainly recent studies on the use of molecular Tp genotyping, neurotropic potential and geographic distribution of subfamilies (Clado).

Inclusion criteria

The following inclusion criteria were used: studies involving molecular typing of Treponema pallidum subsp. pallidum by the MLST technique.

Exclusion Criteria

The following were excluded: meta-analyses, epidemiological, therapeutic studies that addressed other Tp subspecies, other pathogens or that addressed other issues that were not related to syphilis.

Fifty-six articles were identified in the following databases: 0 in Scielo, 9 in Pubmed, 0 in LILACS, 1 in Clinical Trials, 15 in Embase, 31 in the Cochrane Library and 0 in the CAPES Theses and Dissertations Catalog.

Of these 56 articles, 7 were selected for full reading, and 49 were excluded after reading the title and abstract according to Flowchart 1.

The main reasons for excluding articles were: 2 meta-analysis studies, 1 epidemiological study, 1 therapeutic study, 1 described the development of the PubMLST platform, 4 articles talked about treponema pallidum subsp. perternue, 1 article addressed Treponema pallidum subsp. endemicum , 3 articles addressed other issues unrelated to syphilis , 8 duplicate articles and 28 addressed other pathogens.

It was observed that the 7 selected articles included a total of 574 patients and 1,169 biological samples that corresponded to the inclusion criteria proposed in this review. Of these 1,169 samples, 199 of blood, 522 of swab,4 of tissue and 50 of CSF and 2 articles do not describe the types of samples analyzed

Most of the bacteria that cause diseases are isolated through laboratory cultivation but only in 2018 Edmondson et al were able to multiply and maintain Tp in the laboratory through a tissue culture system, preserving its morphological and infectious properties for a long period, effectively, but this promising technique is not yet available for most research and diagnostic centers. Further refinement of this long-term subculture system is expected to facilitate the study of the physiological, genetic, pathological, immunological and antimicrobial susceptibility properties of T. pallidum subsp. pallidum and closely related pathogenic species and subspecies of Treponema.

Molecular biology has shown to be effective for the detection and genotyping of Tp, the polymerase chain reaction has high sensitivity and specificity (17), as well as the most modern techniques of genetic sequencing such as MLST (5).

Studies related to the use of MLST may bring important clinical and epidemiological considerations. Six of the seven articles included in this review reported that the presence of the A2058G or A2059G mutation in 23S rRNA, found in samples from the SS14 clade is commonly associated with macrolide resistance (18, 19). In China, mutations associated with macrolide resistance probably occurred as a result of the indiscriminate and excessive use of this class of antibiotics to treat respiratory tract infections and sexually transmitted infections and not by dissemination arising from mutant strains from just a few individuals (9 ), this conclusion is based on the fact that macrolides are rarely prescribed for the treatment of syphilis (20).

Of the 7 articles included in the study, only two performed CSF analysis. The results describe that patients with the 1.1.2 allelic profile, discriminated by the MLST technique, correspond to the 14d/f subtype described by the Enhanced Centers for Disease Control technique in Prevention Typing (ECDCT), thus, the 1.1.2 alleles (14d/ f) were more likely to develop neurosyphilis. Unlike patients with allelic profile 1.3.1 (14d/g) they had a lower propensity to develop the disease. In addition, the authors report that the two main genes of clinical relevance related to neurosyphilis are Tp0548 type f and Tp0705 type, 2 as patients with clinical/laboratory reports compatible with neurosyphilis had these genes in their clinical sample. In clinical practice, if these two genes were detected from the laboratory analysis of the patient's blood sample, the patient would benefit from not needing a lumbar puncture (10).

Liu et al. (2021) studying the Chinese population of Xiamen, observed that the allelic profile most commonly found in biological samples containing Tp was 1.1.8, but did not relate it to NS, despite using CSF in their study. He also mentions that in other countries, the most common allelic profile is 1.1.3, suggesting that the strains tend to remain restricted to certain geographic areas, as it has not yet been introduced in China.

The article published by Liu et al. (2021) is the only one that addresses the use of CSF samples for Tp genotyping using the MLST technique. In this way, we have an excellent tool and great potential for the study of neurosyphilis, which will allow the detection of potentially neurotropic strains, which present greater severity, morbidity and harm to patients.

It is concluded that the determination of the allelic profile through MLST is a high resolution genotyping tool that allows the distinction of subspecies and species of Treponema pallidum, and can be used to differentiate strains within each phylogenetic tree of these microorganisms.

In Brazil, no studies related to Tp genotyping were found.

The mutation most associated with macrolide resistance is A2058G, which is commonly found in Clade SS14-like samples, in addition, the most studied gene to assess macrolide resistance is 23s rRNA.

The MLST technique presents a great perspective for the deepening of questions related to the study of different strains of Tp, especially the characterization of neurotropic strains that cause NS, antibiotic resistance, phylogeny and geographic distribution.

PCDT- Clinical protocols and therapeutic guidelines

Tp- Treponema pallidum subesp . pallidum

MLST- Multilocus Sequence Typing

loci- housekeeping

ST- Sequence typing

NS- Neurosyphilis

CNS- Central Nervous System

CSF-VDRL - Veneral Disease Research Laboratory in the cerebrospinal fluid

CAPES - Theses and Dissertations Catalog

CSF - Cerebrospinal fluid

ECDCT- Enhanced Centers for Disease Control and Prevention Typing

a. Funding: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

b. Competing Interests: The authors have no financial, commercial, political, academic and personal conflicts of interest.

c. Data Availability: Not applicable.

d. Code availability: Not applicable.

e. Ethics approval: This is a review study. The Research Ethics Committee (HUGG) has confirmed that no ethical approval is required.

f. Author Contributions: All authors contributed to the conception and design of the study. Material preparation, data collection and analysis were performed by Beatriz Azevedo and Isabelle Rangel. The first draft of the manuscript was written by Isabelle Rngel and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

g. Consent to participate: Not applicable.

h. Consent for publication: Not applicable.

1-.World Health Organization 2016. Report on global sexually transmitted infection surveillance 2015. WHO Document Production Services, Geneva, Switzerland.

2- GBD 2016 Disease Injury Incidence and Prevalence Collaborators 2017. Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet 390:1211–1259. doi:10.1016/S0140-6736(17)32154-2.

3- Schaudinn F, Hoffman E. 1905. Vorläufiger Bericht über das Vorkommen für Spirochaeten in syphilitischen Krankheitsprodukten und bei Papillomen . Arb Gesundh AMT Berlim 22 :528–534.

4 - Schaudinn F, Hoffmann E. 1905. Über Spirochaetenbefunde im Lymphdrüsensaft Syphilitischer . Dtsch med Wochenschr 31 :711–714. doi:10.1055/s-0029-1188114.

5-GRILLOVÁ L, et al. Molecular characterization of Treponema pallidum subsp. pallidum in Switzerland and France with a new multilocus sequence typing scheme. PLoS One, 13(7), e0200773, Jul. 2018a.

6- MAIDEN, J. C. M. Multilocus Sequence Typing of Bacteria.Annual Review of Microbiology, p. 561-588, 2006 FERNÁNDEZ-NAVAL C, et al,. Multilocus sequence typing of Treponema pallidum subsp. pallidum in Barcelona. Future Microbiol,16:967-976, Sep. 2021.

7- FERNÁNDEZ-NAVAL C, et al,. Multilocus sequence typing of Treponema pallidum subsp. pallidum in Barcelona. Future Microbiol,16:967-976, Sep. 2021

8- Liu D, He SM, Zhu XZ, Liu LL, Lin LR, Niu JJ, Yang TC. Molecular Characterization Based on MLST and ECDC Typing Schemes and Antibiotic Resistance Analyses of Treponema pallidum subsp. pallidum in Xiamen, China. Front Cell Infect Microbiol. 2021 Feb 19;10:618747. doi: 10.3389/fcimb.2020.618747

9- Sahi SK, Zahlan JM, Tantalo LC, Marra CM. A Comparison of Treponema pallidum Subspecies pallidum Molecular Typing Systems: Multilocus Sequence Typing vs. Enhanced Centers for Disease Control and Prevention Typing. Sex Transm Dis. 2021 Sep 1;48(9):670-674. doi: 10.1097/OLQ.0000000000001378. PMID: 34110749; PMCID: PMC8373628.

10-LIU D, et al. Molecular Characterization Based on MLST and ECDC Typing Schemes and Antibiotic Resistance Analyses of Treponema pallidum subsp. pallidum in Xiamen, China. Front Cell Infect Microbiol, 10:618747. Feb. 2019

11- POSPÍŠILOVÁ P, GRANGE PA, GRILLOVÁ L, MIKALOVÁ L, MARTINET P, JANIER M, et al. Multi-locus sequence typing of Treponema pallidum subsp. pallidum present in clinical samples from France: Infecting treponemes are genetically diverse and belong to 18 allelic profiles. PLoS ONE, 13, e0201068, 2018

12- GRILLOVÁ L, et al. Multilocus sequence typing of Treponema pallidum subsp. pallidum in Cuba from 2012 to 2017. J Infect Dis, 2018b

13- VRBOVÁ E.; GRILLOVÁ, L.; MIKALOVÁ, L.; POSPÍŠILOVÁ, P.; STRNADEL, R.; DASTYCHOVÁ, E.; KOJANOVÁ, M.; KREIDLOVÁ, M.; VAŇOUSOVÁ, D.; ROB, F.; PROCHÁZKA, P.; KRCHŇÁKOVÁ, A.; VAŠKŮ V.; WOZNICOVÁ, V.; HEROLDOVÁ, M.D.; KUKLOVÁ, I.; ZÁKOUCKÁ, H.; ŠMAJS, D.; MLST typing of Treponema pallidum subsp. pallidum in the Czech Republic during 2004-2017: Clinical isolates belonged to 25 allelic profiles and harbored 8 novel allelic variants. PLoS One. v. 14(5), e0217611, Maio 2019

14- MARRA CM, et al. Enhanced molecular typing of treponema pallidum: geographical distribution of strain types and association with neurosyphilis. J Infect Dis, 202(9) pp. 1380-8.2010, Nov. 2010

15- Nechvátal L, Pětrošová H, Grillová L, Pospíšilová P, Mikalová L, Strnadel R, Kuklová I, Kojanová M, Kreidlová M, Vaňousová D, Procházka P, Zákoucká H, Krchňáková A, Smajs D. Syphilis-causing strains belong to separate SS14-like or Nichols-like groups as defined by multilocus analysis of 19 Treponema pallidum strains. Int J Med Microbiol. 2014 Jul;304(5-6):645-53. doi: 10.1016/j.ijmm.2014.04.007

16- LAFOND RE, LUKEHART SA. Biological basis for syphilis. Clin Microb Reviews, pp. 29-49, 2006

17- Zhou P, Li K, Lu H, Qian Y, Gu X, Gong W, Tucker JD, Cohen MS. Azithromycin treatment failure among primary and secondary syphilis patients in Shanghai. Sex Transm Dis. 2010 Nov;37(11):726-9. doi: 10.1097/OLQ.0b013e3181e2c753. PMID: 20644500; PMCID: PMC3114640.

18- Molini BJ, Tantalo LC, Sahi SK, Rodriguez VI, Brandt SL, Fernandez MC, Godornes CB, Marra CM, Lukehart SA. Macrolide Resistance in Treponema pallidum Correlates With 23S rDNA Mutations in Recently Isolated Clinical Strains. Sex Transm Dis. 2016 Sep;43(9):579-83. doi: 10.1097/OLQ.0000000000000486. PMID: 27513385; PMCID: PMC4982755.

19- Lu H, Li K, Gong W, Yan L, Gu X, Chai Z, Guan Z, Zhou P. High frequency of the 23S rRNA A2058G mutation of Treponema pallidum in Shanghai is associated with a current strategy for the treatment of syphilis. Emerg Microbes Infect. 2015 Feb;4(2):e10. doi: 10.1038/emi.2015.10

20- Edmondson DG, Hu B, Norris SJ. Long-Term In Vitro Culture of the Syphilis Spirochete Treponema pallidum subsp. pallidum. mBio. 2018 Jun 26;9(3):e01153-18. doi: 10.1128/mBio.01153-18. PMID: 29946052; PMCID: PMC6020297.

No competing interests reported.

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Use of the MLST technique to describe the allelic profile of Treponema pallidum in CSF samples: a systematic review

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