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.During the last 4 to 5 years, genetic variation in Mycobacterium leprae has been investigated for the purpose of strain typing. Although the M. leprae genome (4) has undergone reductive evolution and is highly mutated, limited genome variability has been found between global isolates, and except for loci prone to mutation, such as variable-number tandem repeats (VNTR) (7,8,12,13,16,19,22,24,25,26), M. leprae strains are highly clonal species. Three single nucleotide polymorphisms (SNPs) were subsequently discovered by further genome sequencing efforts that allowed the separation of global isolates into four subtypes (14).Formal, systematic study of M. leprae diversity by the application of the known polymorphic markers in defined endemic settings for studying extant population structures and leprosy transmission is limited. Previously, we presented the outcome of a focused study in Qiubei County in Yunnan Province, South West China (22). Using VNTR loci, we discovered subgroups within a major lineage. A differential geographical distribution of these subgroups was seen across the county. Furthermore, we noted the conservation of M. leprae genotypes carried by patients of multicase families (MCFs), which is indicative of localized transmission from shared sources.We now extend such approaches to Cebu, Philippines, an island in the Central Visayas where leprosy is still in existence. From a case detection rate of 5.1 in 2001 to the current rat...

Real-Time PCR and High-Resolution Melt Analysis for Rapid Detection of Mycobacterium leprae Drug Resistance Mutations and Strain Types

“…There is only one report for this mutation type (24), which incidentally was detected in the same studied population, i.e., leprosy patients in Cebu, Philippines. The identification of multiple cases with this mutation in our study sample was due to transmission within a closely linked community, as captured and described in greater detail by Li et al by prospective molecular epidemiological approaches (18,30,31). Singh et al recently reported a method based on TaqMan probe assays for rpoB and folP1 (35).…”

“…HRM assays that can be designed based on principles as described herein for the discrimination of the 16 subtypes were beyond the scope of the present study goals and are also restricted by the availability of a representative collection of all strain subtypes. Furthermore, to date, the highest resolution of strains within SNP subtypes has been achieved by VNTR strain typing (7,15,30,31).…”

“…In the interim, the real-time PCR-HRM assays described here are viable, simple options and can be easily integrated into practice by centralization of tests in a reference laboratory. a The PCR-RFLP assay was performed as previously described (31). b The NHDP63 allele is assigned to cluster 1 and the alternative allele to cluster 2. c V, HRM automatically grouped the three strains NP106, NP113, and 125 into a different cluster (variant).…”

“…The clinical skin biopsy samples used in this study were obtained from patients presenting at the Cebu Skin Clinic, Leonard Wood Memorial Leprosy Research Centre, Philippines, as previously described (n ϭ 121) (18,31) and at the Anandaban Hospital, Kathmandu, Nepal (n ϭ 25; collected during 2000 to 2010). The Philippine samples were stored in 70% ethanol at the time of collection.…”

DNA Fingerprinting of <em>Mycobacterium leprae</em> Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)

“…Multiple locus VNTR analysis (MLVA) 10 has been used to study leprosy evolution and transmission in several countries including China 11,12 , Malawi 8 , the Philippines 10,13 , and Brazil 14 . MLVA involves multiple steps.…”