Multilocus microsatellite typing (MLMT) reveals high genetic diversity of Leishmania infantum strains causing tegumentary leishmaniasis in northern Italy

This study utilizes multilocus microsatellite typing to reveal high genetic diversity among *Leishmania infantum* strains causing tegumentary leishmaniasis in northern Italy, identifying a distinct parasite population exclusive to skin infections that underscores the necessity of integrated One Health surveillance to understand the disease's epidemiology.

The Gist

Imagine the parasite Leishmania infantum as a master of disguise. Usually, we think of this parasite as a "systemic invader" that hides in the body's deep organs (like the liver and spleen), causing a severe illness called Visceral Leishmaniasis (VL). But recently, in Northern Italy, this same parasite has been showing up in a different costume: causing skin sores and mucosal issues, a condition called Tegumentary Leishmaniasis (TL).

This paper is like a high-tech detective story where scientists used a genetic "fingerprinting" tool to figure out: Are these skin-causing parasites the same "family" as the organ-causing ones, or are they a completely different gang?

Here is the breakdown of their findings using simple analogies:

1. The Detective Tool: Genetic Fingerprints

The researchers didn't just look at the parasites under a microscope; they used a technique called Multilocus Microsatellite Typing (MLMT).

• The Analogy: Imagine trying to identify people in a crowded room. You could just look at their height (which might be similar for everyone), or you could ask them to recite a specific, complex rhyme that only they know.
• The Science: The scientists looked at 15 specific "rhymes" (microsatellite markers) in the parasite's DNA. Because these rhymes vary wildly between individuals, they act like unique fingerprints. This allowed the team to distinguish between 44 different skin-infection cases with incredible precision.

2. The Big Discovery: Three Distinct "Tribes"

When the scientists compared the fingerprints of the skin parasites against those from organ infections (VL), dogs, and sand flies, they didn't find a messy mix. Instead, they found three distinct genetic "tribes" or populations that rarely mix:

• Tribe A (The Dog Gang): This group is mostly found in dogs. It's the classic, widespread version of the parasite in Italy. Interestingly, it almost never infects humans in this specific region. It's like a dog-specific club that humans just don't get invited to.
• Tribe B (The Mixed Bag): This tribe is a bit of a chameleon. It infects both people with severe organ disease (VL) and some people with skin disease (TL). It also shows up in sand flies. This suggests that having this specific genetic "background" doesn't guarantee you'll get a specific type of disease; other factors (like the person's immune system) decide the outcome.
• Tribe C (The Skin Specialists): This is the big news. The researchers found a group of parasites that only showed up in people with skin or mucosal infections. They were never found in dogs or in people with organ disease in this area.
  • The Metaphor: Think of Tribe C as a specialized delivery service that only delivers packages to the front door (the skin) and refuses to go inside the house (the organs). They are a hidden lineage that was previously invisible because scientists weren't looking at skin cases closely enough.

3. The "One Health" Puzzle

The study emphasizes a concept called One Health, which is like realizing that to solve a mystery, you need to talk to the homeowner, the pet, and the delivery driver (the sand fly) all at once.

• By looking at humans, dogs, and insects together, the scientists realized that the parasites causing skin disease in Italy are genetically distinct from the ones causing organ disease.
• They found that the "Skin Specialists" (Tribe C) are so different that they form their own unique branch on the family tree, separate from the "Organ Invaders" and the "Dog Specialists."

4. Why Does This Matter?

• Hidden Diversity: For a long time, scientists thought L. infantum was mostly one thing. This study shows it's actually a complex family with different "cousins" that prefer different neighborhoods in the body.
• Better Surveillance: If we only watch dogs or only watch people with organ disease, we miss the "Skin Specialists." To stop the spread of the disease, health officials need to monitor all three groups (humans, animals, and insects) simultaneously.
• Evolution in Action: The fact that these tribes are so different suggests they are evolving on separate paths. It's possible that Tribe C is adapting specifically to survive in the skin, perhaps because of how the sand fly bites or how the human immune system reacts.

The Bottom Line

This paper reveals that the parasite causing skin sores in Northern Italy isn't just a "mild version" of the one causing organ failure. It's often a genetically distinct group that has carved out its own niche. By using advanced genetic fingerprinting, the researchers uncovered a hidden world of parasite diversity, proving that to truly understand and fight this disease, we need to look at the whole picture: the dog, the fly, and the human, all together.

Technical Summary

1. Problem Statement

• Context: Leishmania infantum is traditionally associated with Visceral Leishmaniasis (VL), a systemic and often fatal disease. However, in southern Europe (specifically northern Italy), there has been a re-emergence of Tegumentary Leishmaniasis (TL), which includes Cutaneous (CL) and Mucosal (ML) forms.
• Knowledge Gap: While L. infantum is known to cause both VL and TL, the genetic basis for this tissue tropism (why some strains cause systemic disease while others remain localized to the skin/mucosa) is poorly understood. Previous studies in the Emilia-Romagna (E-R) region suggested the presence of hybrid strains, but the population structure of TL-specific strains remained unclear.
• Objective: To investigate the genetic diversity and population structure of L. infantum strains causing TL in northern Italy and compare them with sympatric strains from VL cases, dogs (the primary reservoir), and sand fly vectors.

2. Methodology

• Study Design: A One Health approach integrating data from humans (TL and VL), dogs (CanL), and vectors (sand flies) from the Emilia-Romagna region (2013–2024).
• Sample Collection:
  • Total Strains: 133 L. infantum strains.
  • New Data: 44 TL strains (39 CL, 5 ML) obtained from biopsies (FFPE and fresh) and cultured isolates.
  • Comparative Data: 52 canine isolates, 12 sand fly strains, and 25 human VL strains (some from previous studies, some newly analyzed to reduce sampling bias).
• Molecular Techniques:
  • Species Confirmation: PCR and sequencing of ITS-1 or Hsp70 genes confirmed all samples as L. infantum.
  • Genotyping: Multilocus Microsatellite Typing (MLMT) using 15 polymorphic dinucleotide markers (e.g., Li41-56, Lm2TG, Lm4TA).
  • Data Processing: Microsatellite repeat numbers were determined relative to a standard strain (MHOM/TN/1980/IPT1) to generate Multilocus Genotypes (MLGs).
• Statistical & Population Genetic Analysis:
  • Clonal Correction: Unique MLGs were retained to minimize bias from clonal expansion (final dataset $N=106$ for structure analysis).
  • Clustering: Bayesian clustering (STRUCTURE v2.3.4) and Discriminant Analysis of Principal Components (DAPC) were used to infer population structure.
  • Genetic Metrics: Calculated allelic diversity ($A$, $MNA$), heterozygosity ($H_e$, $H_o$), inbreeding coefficients ($F_{is}$), and Hardy-Weinberg equilibrium (HWE).
  • Differentiation: Pairwise fixation indices ($F_{st}$) were calculated to measure genetic differentiation between populations.
  • Phylogenetics: A microsatellite-based Bayesian tree was constructed using the BEAST2 package with the Sainudiin mutation model.

3. Key Results

• High Genetic Diversity: Among the 44 TL strains, 43 distinct MLGs were identified, indicating high genetic polymorphism. Only two strains (ID6 and ID17) shared an identical MLG.
• Population Structure (Three Distinct Clusters): Analysis consistently identified three highly differentiated genetic populations ($F_{st} > 0.25$):
  • PopA: Primarily composed of canine isolates (40/41 strains) and one TL strain from outside the study region (Apulia). This population corresponds to the widespread L. infantum MON-1 zymodeme. Crucially, PopA showed no overlap with local TL cases in Emilia-Romagna.
  • PopB: A mixed population containing all VL strains, 9 TL strains, and 5 sand fly strains. This suggests a lineage capable of causing both systemic and tegumentary disease.
  • PopC: A population found exclusively among TL cases (28/44 strains) and one sand fly strain. This represents a distinct genetic lineage associated specifically with tegumentary manifestations in this region.
• Genetic Differentiation:
  • Pairwise $F_{st}$ values were very high between all groups (PopA vs. PopB: 0.61; PopA vs. PopC: 0.58; PopB vs. PopC: 0.48), confirming strong genetic separation.
  • Heterozygosity Patterns:
    • PopA and PopB: Showed positive $F_{is}$ values (deficit of heterozygotes), indicative of clonal expansion and inbreeding.
    • PopC: Showed a negative $F_{is}$ (-0.197), indicating an excess of heterozygotes. This suggests distinct evolutionary dynamics, potentially involving weaker bottlenecks or different reproductive strategies compared to VL-associated strains.
• Spatial and Temporal Distribution: The distribution of PopB and PopC within the Emilia-Romagna region was not geographically or temporally clustered, suggesting these populations co-circulate in the same endemic area.

4. Key Contributions

• Discovery of a TL-Specific Lineage: The study identifies PopC, a genetic population of L. infantum that is strongly associated with tegumentary leishmaniasis and distinct from the populations causing visceral disease or circulating in local dogs.
• Decoupling of Host and Strain: The findings challenge the assumption that local dog populations are the direct source of human TL cases in this region, as the dominant canine population (PopA) does not overlap with the human TL population (PopC).
• Evidence of Hybrid/Admixed Strains: The presence of admixed strains (e.g., PopB/C) and the distinct placement of TL strains in phylogenetic trees suggest dynamic genetic exchange and potentially hybrid origins (consistent with previous Hsp70 sequencing data).
• Methodological Validation: Demonstrates the utility of MLMT combined with Bayesian and multivariate approaches to resolve complex population structures in Leishmania that are not apparent with single-gene typing.

5. Significance and Implications

• Epidemiological Insight: The existence of a distinct TL-associated population (PopC) implies that the re-emergence of cutaneous leishmaniasis in northern Italy is driven by specific parasite lineages, not just a general increase in transmission of the standard VL strain.
• One Health Surveillance: The study underscores the necessity of integrated surveillance that includes human, animal, and vector data. Relying solely on canine reservoir data (PopA) would miss the specific drivers of human TL (PopC).
• Pathogenesis: The distinct genetic background of PopC and its unique heterozygosity profile suggest that parasite genetics play a significant role in determining tissue tropism, although host factors remain critical.
• Public Health Strategy: Understanding these distinct lineages is crucial for developing targeted control measures, diagnostics, and potentially vaccines, as the "standard" L. infantum models may not represent the strains causing the re-emerging skin disease.

In conclusion, this paper provides robust evidence that L. infantum in northern Italy is not a monolithic population but consists of highly differentiated lineages with distinct epidemiological and clinical associations, necessitating a shift in how leishmaniasis surveillance and control are approached in the region.