All isolates were collected in Guangxi region of southern China on the southeastern corner of the Yunnan-Guizhou Plateau, situated from 20.54°N to 26.23°N and from 104.08°E to 112.04°E. This region borders Vietnam to the southwest and is surrounded by Guangdong, Guizhou, Yunnan, and Hunan Provinces in China. The region has a terraced topography sloping from the northwest to the southeast, with hilly land constituting 85% of its total area and plains constituting 15%. The region has a subtropical humid monsoon climate, with average daily temperatures of 16°C–23°C. The rainy season lasts from April until September, with an annual rainfall of 1,500 mm–2,000 mm.

Farmers trapped 43 adult hoary bamboo rats (R. pruinosus) from 8 different districts across Guangxi Province. The 15 female and 28 male captured rats were euthanized and aseptically dissected as described (7). P. marneffei was recovered from the main organs of the rats (lungs, liver, and spleen) by injection onto Sabouraud dextrose agar and brain–heart infusion agar and cultured at 25°C and 37°C, respectively, for 3–4 weeks. Both media were supplemented with chloramphenicol (0.05 mg/mL). Species identification of P. marneffei was based on conversion of yeast to hyphae at 25°C, secretion of a characteristic bright red pigment, and morphologic identification of colonies and conidia formation. In addition to the rodent isolates, 40 isolates were collected from human patients (including 36 persons positive for HIV) across Guangxi Province. The clinical specimens included blood, skin biopsy samples, pus from subcutaneous abscesses, lymph node biopsy samples, and bronchoalveolar lavage pellets. The linear geographic distance among the sites ranged from 133 km to 503 km, with an average distance of 224 km (Table 1; Figure 1).

DNA was extracted from 7-day-old cultures of each P. marneffei isolate as described (8). Six microsatellite-containing loci were chosen from the panel described by Fisher et al (9). These loci (PM5, PM6, PM19, PM22, and PM23) were selected because of their high discriminatory power within a previously genotyped cohort of isolates obtained from humans and bamboo rats in Thailand (10); the loci vary in the length of the microsatellite-containing repeat region. The 6 loci were amplified according to published PCR protocols (9). Subsequently, the PCR products were subjected to electrophoresis through a capillary sequencer with a POP6 gel and a ROX-500 internal size standard (Applied Biosystems, Foster City, CA, USA). Alleles were scored by using Genotyper software (Applied Biosystems), and multilocus genotypes for each isolate were then generated by scoring length polymorphisms at the 6 microsatellite-containing loci. Microsatellite types were subsequently identified for newly typed isolates by comparing this novel dataset from China against previously genotyped isolates from Thailand (10).

Multilocus microsatellite types (MLMTs) were manipulated and analyzed by using the GenAlEx software add-in for Excel (11). The 86 Chinese isolates were coded into 2 populations according to host, human or rat, and were compared against 186 P. marneffei MLMTs from Thailand, also coded into 2 populations according to host. Isolates were further partitioned into 2 broad geographic regions, China or Thailand. Basic data exploration was undertaken to calculate allele and genotype frequencies and to calculate diversity statistics. The number of identical genotypes shared between hosts (human and rat) was then determined for each region by assessing which MLMTs were common to both host species. Subsequently, the genetic distances between the MLMT genotypes of P. marneffei from different hosts were calculated and visualized by using the neighbor-joining tree algorithm in GenAlEx (11).

The distribution of genetic variation across Southeast Asia between isolates of P. marneffei was estimated by performing an analysis of molecular variance (AMOVA) (12). AMOVA is a statistical technique that estimates the extent of genetic differentiation between individuals and populations directly from molecular data. The technique treats the raw molecular data as a pairwise matrix of genetic distances between all possible combinations of P. marneffei isolates, with submatrices corresponding to the different hierarchical data partitions (here, the genetic differences between P. marneffei infecting different host individuals, host species, and geographic regions). The data are then analyzed within a nested analysis of variance framework. Means squares are computed for each hierarchy of data, enabling significance testing between the following: 1) individual P. marneffei genotypes within hosts; 2) genotypes distributed between hosts (humans and bamboo rats); and 3) genotypes distributed between region (China and Thailand). Randomized distributions of the data are generated through random permutations, and the rejection of the null hypothesis (Ho = no significant component of variation occurs between the hierarchical divisions) then demonstrates the existence of population subdivision, either at the level of geography or host.

Subsequently, the presence of fine-scale geographic substructure within regions was determined by the use of Mantel tests. Mantel tests work by creating 2 pairwise matrices from each collection of isolates corresponding to 1) the pairwise genetic distances between isolates, and 2) the pairwise spatial distances between isolates in kilometers. The observed correlation between these genetic and geographic distances within China and for each host population (human and rat) were calculated and compared against 1,000 randomized datasets. The observed correlations were then considered significant (>0) if they exceeded 950 of the randomized datasets.

Our survey demonstrated that 100% of the 43 adult R. pruinosus rats captured in Guangxi Province were positive for P. marneffei (Table 1). All the P. marneffei–positive R. pruinosus rats appeared healthy and, at necropsy, no visible pathologic changes were observed in any of the internal organs of the rats. The strains were most frequently isolated from lung, liver, and spleen tissues; however, no isolate was recovered from the embryonic tissue of pregnant rats (n = 15). This finding suggests that vertical transmission of infection within R. pruinosis rats does not occur. Forty isolates were obtained from human, including 36 patients who were positive for HIV. The strains were most frequently recovered from blood cultures (100%), followed by bone marrow aspirate (91%), skin biopsy specimens and pus of subcutaneous abscess (84%), lymph node biopsy specimens (34%), and bronchoalveolar lavage pellets (2.5%).

A single PCR amplification product was observed for all 83 isolates at each locus. Because P. marneffi has a haploid genome, this finding suggests that none of the cultures were composed of a mixture of P. marneffei strains and that hosts were therefore infected with single-genotype infections. All 6 MLMTs were polymorphic; 65 alleles were found in the pooled P. marneffei populations (human and rat), ranging from 3 alleles for locus PM25 to 11 for locus PM5 (Table 2). The numbers of alleles, haploid gene diversity, and distribution of private alleles were significantly different between human- and rat-associated isolates of P. marneffei in Guangxi (Table 2). Human-associated P. marneffei isolates in Guangxi were more polymorphic (Table 3) and showed higher haploid genetic diversity (Table 3) and numbers of unique alleles (Table 3).

MLMT analyses of the 83 isolates from Guangxi recovered 59 distinct multilocus microsatellite barcode types (MTs). A total of 38 MTs were found infecting humans, and 22 were infecting bamboo rats. A single MT haplotype was found to co-occur in both human and rats (Figure 2). The probability of observing infections with identical genotypes in different hosts by chance alone can be approximated as ≈5.46 × 10⁶; this occurrence is therefore statistically highly unlikely to occur, and we conclude that these 2 hosts were co-infected from a single clonally reproducing individual of P. marneffei. Although the bamboo rats in question were trapped in Hezhou, the human isolate was recovered from Hechi, 355 km distant; this observation could indicate either that the patient traveled to and acquired infection in the environs of Hezhou, or infectious P. marneffei conidia can disperse over that physical distance.

We then tested whether P. marneffei isolates from humans and rats represented genetically unique subpopulations of the total genetic diversity by using AMOVA (Table 4). Although isolates recovered from Thailand and China are clearly genetically isolated populations, only 2% of the total diversity was partitioned between host species within these regions. Within Guangxi, however, this minor component of diversity was significant (p = 0.008), which suggests that humans and bamboo rats in Guangxi are not infected by P. marneffei in a completely random manner and that some underlying genetic structure exists.

To test whether this component of variation between humans and bamboo rats was related to spatial factors, we tested the extent of correlation between geographic distance and genetic distance across 3 components of P. marneffei diversity in Guangxi by using Mantel tests for the following data partitions: 1) human and bamboo rat isolates together; 2) human isolates alone; and 3) bamboo rat isolates alone. Combined human and bamboo rat data showed a significant spatial component to the distribution of genetic diversity across Guangxi (p = 0.001). However, this effect is not observed for isolates collected from humans (p = 0.323) but is observed for isolates collected from bamboo rats (p = 0.001). This result shows that the spatial signal in the dataset is attributable to the rodent-associated isolates.

The finding that rodent isolates are more spatially structured than human-associated isolates is confirmed by assessing the proportion of identical genotypes that are to be found infecting either human or rodent hosts within the different sample sites in Guangxi (Figure 2). This analysis showed that although 9 MLMT haplotypes infected >1 bamboo rat within a sample site, only a single MLMT haplotype was shared among human patients within 1 sample site (Hezhou). That no MTs were shared by bamboo rats between trapping sites is strong evidence that P. marneffei genotypes are spatially patchy within Guangi Province. Furthermore, although no MLMT haplotypes were shared between bamboo rats from different sample sites, 2 MLMT haplotypes (Human18, Guigang and Human32, Nanning; Human22, Hechi and Rat29, 30, 34, and 35, Hezhou) were shared between humans or bamboo rats from different sample sites. Together, these data show that humans are exposed to a greater diversity of P. marneffei genotypes relative to the bamboo rats, despite occupying a similar geographic region.