Leprosy in Philippines and in the island province of Cebu.
In 1998, about a decade after multidrug therapy (MDT) was implemented nationwide, the Philippines reached its elimination goal, with a prevalence of 0.9 cases/10,000 persons, with a total of 6,872 registered cases. After another decade of leprosy elimination campaigns, this has further gone down to a prevalence of 0.36 cases/10,000 persons, with a total of 3,156 registered cases. For Cebu, data from the 2001-2006 period show that the prevalence has been sustained: 0.48 cases/10,000 persons in 2001 and 0.46 cases/10,000 persons in 2006.
The CSC diagnoses around 200 patients a year, more than half of the new cases from the island. The numbers of patients according to the Ridley-Jopling clinical classification are shown in Table . These patients come from all parts of the island province, voluntarily or as referrals. A large proportion of data from CSC cases since 2003 are included in the current study.
New leprosy cases diagnosed at CSC, LWM, 2000 to 2007
Cebu is a narrow island, 225 km long and 25 km across at the widest point (Fig. ). Metro Cebu comprises seven cities (Carcar City, Cebu City, Danao City, Lapu-Lapu City, Mandaue City, Naga City, and Talisay City) and six other municipalities on the east. Each city is administratively divided into barangays (akin to a county or village). Cebu City per se is located centrally within the Metro Cebu area, while Lapu-Lapu City is in Mactan Island and is connected to the main island by a bridge. The CSC is in Cebu City, while the leprosarium is located in Mandaue City. It is slightly over 10 km north of the CSC. Of the 228 patients in this study, one-third reside in Cebu City (Fig. ).
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 (more ...)
The majority of the patients were bacteriological index (BI) positive, as judged by microscopic examination of slit skin smear (SSS) samples or biopsy specimens and were prescribed the standard multibacillary WHO MDT treatment (1-year duration in Philippines). Considering the “L” and “M” groups (n = 178), the ages of the patients ranged from 5 to 71 years, with a mean of 30 years. The proportions of patients under 30 and 40 years of age were 66% and 80%, respectively. The ratio of male to female patients was 3:1.
MLVA-based genomic variation of M. leprae in patients in Cebu, Philippines.
Skin biopsy specimens taken from leprosy patients at the time of diagnostic procedures or during treatment (as in the case of 38 of the 100 M group samples) were the source of M. leprae
DNA in these studies. Multiple-locus VNTR analysis (MLVA) was performed for a total of 228 specimens using multiplex PCR and FLA procedures described previously (8
) to determine the number of repeats (hereafter known as alleles). The samples from patients (n
= 21) with a low BI (BI < 1.5) did not yield data for all loci and were excluded from the subsequent analysis.
The MLVA data for 15 loci from the 207 Cebu samples, NHDP63 reference DNA, and sequenced M. leprae
strain TN (4
) were analyzed for allelic diversity and population structure. The allelic diversity across the 15 loci ranged from 0.1 to 0.94, and the heterozygosity value for the entire data set is 0.5334 (21
Comparison of M. leprae VNTR patterns in SSS samples and skin biopsy specimens.
To compare the stabilities of VNTRs in different body compartments, VNTR data for biopsy specimens and SSS samples from 10 high-BI patients were also analyzed (Table ) and were found to be mostly similar. We noted extra product peaks for the SSS for some of the more variable stutter-prone microsatellites. Such patterns may represent variants of a dominant isolate. SSS is usually a mix of smears from the lesion site (biopsy specimen) and as well as from other body sites.
Comparison of M. leprae VNTR patterns between skin biopsy specimens and pooled SSS samples for 10 patients
Rapid PCR-RFLP SNP subtyping of M. leprae in patients in Cebu, Philippines.
By using the NEBcutter2 program, we were able to identify commercially available enzymes that are suitable for the detection and separation of the four SNP subtypes according to the scheme shown in Fig. and . The recognition sequences for SmlI, BstUI, and CviKI-1 are CTYRAG, RGCY, and CGCG, respectively.
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 , 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 (more ...)
At first, we verified the feasibility of using one, two, or three enzymes for subtyping of SNPs 1 to 4 for reference M. leprae strains as shown in Fig. . The restriction enzyme products could be resolved on a 3% agarose gel for SmlI and BstUI. A novel primer set was designed for the SNP 2 locus to eliminate non-SNP CviKI-1 cutting sites. However, there are still two recognition sites: one from the SNP “T” allele would yield fragments of 61, 11, and 42 bp, while amplicons lacking the SNP “T” allele would produce 72- and 42-bp fragments. The 3% agarose gel system was not adequate to reliably differentiate the 72- and 61-bp CviKI-1 fragments; therefore, a 12% acrylamide gel system was used.
After establishing the PCR-RFLP method for separating the isolates of SNP types 1 to 4, we applied it to all 100 samples from the “M” study. The first step of PCR amplification of SNP locus 3 revealed that 99% of the DNAs yielded products, and 18 samples could be digested with BstUI. One of the samples did not yield PCR products for all the three SNP loci. DNA sequencing of the PCR amplicons of the remaining samples confirmed that all BstUI-sensitive amplicons carried SNP allele “C,” while a random selection of the BstUI-resistant samples carried the “A” allele. In the next step, we examined the susceptibility of SNP locus 1 to SmlI. None of the amplicons were digested, as was corroborated by the sequence of the products, and thus, SNP type 4 M. leprae strains are absent in this clinical sample set. Finally, to classify the strains carrying the SNP locus 3 BstUI-resistant phenotype into type 1 or type 2, CviKI-1 enzyme digestion of the locus 2 PCR products was performed. One sample of type 2 was confirmed by sequencing at SNP loci 2 and 3. Another isolate that was restriction positive was sequenced was confirmed to carry the “T” allele; however, a PCR product for locus 3 was not obtained, so we were unable to classify it as being type 2, 3, or 4. One of the samples gave mixed cut and uncut CviKI-1 RFLP patterns. Furthermore, both alleles G and T were observed in the DNA sequence. Interestingly, short tandem repeat (STR) typing of this DNA (M55) also revealed mixed signals at multiple loci even when independent aliquots of biopsy specimen sections of this sample were tested, indicating a mixed infection or contamination of the specimen. In summary, we were able to SNP subtype 97 of the 100 specimens.
VNTR alleles predictive of SNP type 3 for Philippine isolates.
Correlating the SNP subtypes with the VNTR profiles of 15 loci revealed that the alleles at the (GGT)5 and 21-3 loci were 4 and 1 or 2, respectively, for the SNP type 3 isolates (Fig. ), while other combinations of alleles such as 4:3, 5:3, 5:1, and 5:2 were seen in isolates of SNP types 1 and 2.
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 (more ...)
To determine if this allelic signature is predictive of SNP type 3 in this population, we reviewed the 207 VNTR patterns and identified 14 other SNP 3 candidates (10 L and 4 R samples). Of these, we first tested eight L and four R samples. As controls, we randomly tested other “L” (n = 6) and “R” (n = 1) samples that did not have the VNTR pattern of interest. All eight L samples were of SNP type 3 (Fig. ), while the “controls” were not. The “R” samples had degraded, or very little was remaining at the time when these new assays were being tested, and we could recover PCR amplicons for one sample (SNP type 3). Subsequently, we completed the PCR-RFLP SNP typing of the remaining 64 L samples and demonstrated that the SNP type 3 prediction rule was valid.
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 (more ...) Population structure of M. leprae isolates from Cebu, Philippines.
There are several computation models and software to infer clustering and/or phylogenetic relationships with the molecular data set (1
). We applied MP principles, assumed a stepwise mutation model, and executed the analysis using the PAUP software program (17
). By arbitrarily defining the sequenced strain TN as the outgroup taxon, 50% consensus trees were generated. Since the allelic diversity of each locus was found to be different in this study population, we weighted the loci according to the inverse of the individual allelic diversity index for MP. Neighbor joining and unweighted-pair group method using average linkages were also executed from the same data sets (data not shown) in PAUP.
Five major groups emerged in phylogenetic trees using MP (Fig. ), neighbor joining, and the unweighted-pair group method using average linkage algorithms, which we have named groups A, B, C, D, and T. The T group, comprising 10 isolates, is seen proximal to the sequenced TN strain. This grouping may be attributed to the three copies of the 23-3 VNTR locus that are rare in the entire data set and other alleles shared with strain TN.
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, (more ...)
The majority of the remaining isolates fall within groups A, B, C, and D. Groups A and B are separated from groups C and D due to the (AC)8b locus. Groups A and B have 8 copies of the VNTR, whereas in groups C and D, the allele is 7 copies. Group A is then branched into groups A1 and A2; the (AC)9 allele is 8 or 9 copies, respectively. The 18-8 locus, with alleles of 8 or 4 copies, separates B1 from B2.
Group C, comprising 45 clinical isolates, is characterized by low allele numbers for (AC)8b, (TA)18, (GGT)5, (TTC)21, 21-3, and 18-8 VNTR loci. The dominant alleles for (AC)8b, (GGT)5, and 18-8 VNTR loci are 7, 4, and 7 copies, respectively. The range of alleles for (TA)18, (TTC)21, and 21-3 are 12 to 17, 14 to 20, and 1 to 2 copies, respectively, while in groups A, B, and D, they are 21 to 26, 21 to 32, and 3 copies, respectively (Fig. and ).
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 (more ...)
Group D is characterized by a distinctive six-copy allele of the (GGT)5 locus, and all isolates have 10 repeats at the (AC)8a locus.
The majority of the Cebu M. leprae isolates are of SNP type 1. Of the 207 samples for which VNTR patterns were mapped, 100 M, 78 L, and 5 R samples were tested for SNP types. From these samples, 10 L, 18 M, and 1 R samples were confirmed as being of SNP type 3. In the rest of the “R” samples for which DNA is no longer available for testing, three more carry the 4:1 alleles for (GGT)5:21-3. Therefore, we estimate that 16% (32/207) of the study population are of SNP type 3. Interestingly, in trees that were derived based solely on VNTR alleles, the SNP type 3 isolates converged into group C described above, separating from the remaining isolates at a deep or intermediate branch level. The allele profiles for the SNP type 3 versus SNP type 1 isolates are shown in Fig. . A few non-SNP type 3 (of SNP type 1; n = 13) isolates also group with the SNP type 3 isolates in group C, probably due to their low allele numbers, which is typically seen in SNP type 3 isolates (Fig. ) but not in the majority of the SNP type 1 isolates.
Identification of plausible transmission clusters in Cebu by genotyping.
Exposure to the M. leprae source can occur within and outside the household, such as in the community, place of employment, and place education, etc. The barangay of residence at the time of sampling was known for nearly 200 patients. However, daily, weekly, or monthly commute for work or other reasons is not uncommon for residents in the Metro Cebu region.
The patient population studied included several MCFs. MCFs presenting to the clinic within a period of 4 years of each other serve as a model for recent infections from one to the other or from another common source. The 15-locus VNTR profiles of M. leprae from these patients are shown in Table . In general, the mismatched alleles in the MCFs occurred in the more polymorphic and stutter-prone VNTR loci. In the two pairs family 3 (F3) and F4, there were differences in many loci. Accordingly, four of the six MCF pairs appeared to be close in the consensus tree despite allelic differences at one or more loci (Fig. ). Based on these trends, we tentatively define transmission clusters in Cebu as patients/isolates whose M. leprae VNTR alleles are shared at 12/15 loci (includes all minisatellites) and colocalize in the weighted consensus tree.
VNTR profiles of multicase families in the 207 Cebu patient database
Besides examining the genotypes of M. leprae strains in MCFs, we sorted the VNTR profiles according to barangays. There is not much evidence of geographic segregation of strains according to VNTR or SNP profiles. However, several matched pairs emerged (Table ), which are suitable for formal secondary epidemiological investigations of these cases and an active search for other new cases in these barangays, activities not within the scope of the current study.
Identification of pairs of M. leprae isolates with matching VNTR patterns in various barangays in Cebu