Population-based molecular epidemiology of leprosy in Cebu, Philippines

Abstract

To address the persisting problem of leprosy in Cebu, Philippines, we compiled a database of more than 200 patients who attend an established referral skin clinic. We described the patient characteristics in conventional demographic parameters and also applied multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) and single nucleotide polymorphism (SNP) typing for Mycobacterium leprae in biopsied skin lesion samples. These combined approaches revealed that transmission is ongoing, with the affected including the young Cebuano population under 40 years of age in both crowded cities and rural areas of the island. The emergence of multicase families (MCF) is indicative of infection unconstrained by standard care measures. For the SNPs, we designed a low-cost PCR-restriction fragment length polymorphism typing method. MLVA in M. leprae was highly discriminatory in this population yet could retain broad groups, as defined by the more stable SNPs, implying temporal marker stability suitable for interpreting population structures and evolution. The majority of isolates belong to an Asian lineage (SNP type 1), and the rest belong to a putative postcolonial lineage (SNP type 3). Specific alleles at two VNTR loci, (GGT)5 and 21-3, were highly associated with SNP type 3 in this population. MLVA identified M. leprae genotype associations for patients with known epidemiological links such as in MCFs and in some villages. These methods provide a molecular database and a rational framework for targeted approaches to search and confirm leprosy transmission in various scenarios.

Figures

FIG. 1.

(A) Map of Cebu Province, Philippines, with patients' cities of residence. One-third of the patients in this study population are from Cebu City. R, M, and L represent each of the sample collections, followed in parentheses by the number of patients in this study. (B) Distribution of patients from Cebu City according to the barangay (village) of residence. R, M, and L represent each of the sample collections, followed in parentheses by the number of patients in this study. (Maps adapted from http://www.cebu.gov.ph/ with permission.)

FIG. 1.

(A) Map of Cebu Province, Philippines, with patients' cities of residence. One-third of the patients in this study population are from Cebu City. R, M, and L represent each of the sample collections, followed in parentheses by the number of patients in this study. (B) Distribution of patients from Cebu City according to the barangay (village) of residence. R, M, and L represent each of the sample collections, followed in parentheses by the number of patients in this study. (Maps adapted from http://www.cebu.gov.ph/ with permission.)

FIG. 2.

Schematic of M. leprae SNP subtyping of clinical leprosy samples based on PCR-RFLP. (A) The four major SNP types. The numbers below the SNP loci refer to the nucleotide positions in the sequenced TN strain. (B) Scheme of M. leprae SNP subtyping.

FIG. 3.

PCR-RFLP patterns of four M. leprae reference strains (Thai-53 [T53], 3039/21 [3039], NHDP63 [N63], and BR4923 [BR]). The PCR products of SNP loci 1, 2, and 3 were subjected to enzyme digestion. The digested and undigested PCR products were resolved on agarose (A and C) or acrylamide (B) gels and run in pairs; the digested products for each of the strains are shown first. DNA sizing ladders are shown in the left lane in each gel. The numbers on the right side of each gel refer to the lengths (bp) of the PCR and digestion products.

FIG. 4.

The relationship between the 21-3 VNTR allele and the BstUI cutting pattern for 10 Philippine M. leprae samples is shown in A and B, respectively. (A) BstUI-RFLP gel. (B) Agarose gel showing products of multiplex PCR for four VNTR loci. The 21-3 product is the largest. VNTRs at all loci can be seen within this sample set. (C) SNP types. Samples 63 and 69 are indicated below A and B. N63 is NHDP63 (SNP type 3).

FIG. 5.

The 21-3 and (GGT)5 alleles are indicative of SNP types 1 and 3 in Philippine M. leprae strains. The table indicates the VNTR alleles for 21-3 and (GGT)5 for 10 M. leprae specimens. The gel shows the corresponding SNP locus 3 BstUI cutting patterns for these 10 specimens. N63 is NHDP63 (SNP type 3). The graphs show the allele frequency of these loci for SNP types 1 and 3 for 100 M. leprae samples.

FIG. 6.

M. leprae population structure based on VNTR markers and the dominant genotypes identified in Cebu, Philippines. On the left, a 50% consensus phylogenetic tree generated using the MP algorithm is shown, and the major branches are marked A, B, C, D, and T. The source of each M. leprae isolate is indicated in the three columns to the right: study (R, M, and L) sample, barangay, and city, respectively. The conserved allelic patterns within each branch are indicated at the extreme right. The locus order is (AC)8b, (GTA)9, (GGT)5, (AT)17, 21-3, (AC)9, (AT)15, (AC)8a, 27-5, 6-7, (TA)18, (TTC)21, 18-8, 12-5, and 23-3. Clustering of genotypes within MCFs and/or barangays is seen, represented by codes F1 to F6 (VNTR genotypes are shown in Table 3) and B1 to B8 (as listed in Table 4).

FIG. 7.

Comparison of VNTR patterns of SNP type 3 compared to those of SNP type 1 isolates from Cebu, Philippines. The allelic frequencies (y axis) were plotted against the VNTR alleles (x axis) for the locus indicated on the top of each panel. Solid squares and hollow squares represent SNP type 3 and SNP type 1 isolates, respectively.