Multi-locus sequence typing and phylogenetics of Cryptococcus neoformans AD hybrids.

Hybrid AD strains of the human pathogenic Cryptococcus neoformans species complex have been reported from many parts of the world. However, their origin, diversity, and evolution are incompletely understood. In this study, we analyzed 102 AD hybrid strains representing 21 countries on five continents. For each strain, we obtained its mating type and its allelic sequences at each of the seven loci that have been used for genotyping haploid serotypes A and D strains of the species complex by the Cryptococcus research community. Our results showed that most AD hybrids exhibited loss of heterozygosity at one or more of the seven analyzed loci. Phylogenetic and population genetic analyses of the allelic sequences revealed multiple origins of the hybrids within each continent, dating back to one million years ago in Africa and up to the present in other continents. We found evidence for clonal reproduction and long-distance dispersal of these hybrids in nature. Comparisons with the global haploid serotypes A and D strains identified new alleles and new haploid multi-locus genotypes in AD hybrids, consistent with the presence of yet-to-be discovered genetic diversity in haploid populations of this species complex in nature. Together, our results indicate that AD hybrids can be effectively genotyped using the same multi-locus sequencing type approach as that established for serotypes A and D strains. Our comparisons of the AD hybrids among each other as well as with the global haploid serotypes A and D strains revealed novel genetic diversity as well as evidence for multiple origins and dynamic evolution of these hybrids in nature.

In Vivo Efficacy of Olorofim against Systemic Scedosporiosis and Lomentosporiosis.

Clinically relevant members of the Scedosporium/Pseudallescheria species complex and Lomentospora prolificans are generally resistant against currently available systemic antifungal agents in vitro, and infection due to these species is difficult to treat. We studied the in vivo efficacy of a new fungicidal agent, olorofim (formerly F901318), against scedosporiosis and lomentosporiosis in neutropenic animals. Cyclophosphamide-immunosuppressed CD-1 mice infected by Scedosporium apiospermum, Pseudallescheria boydii (Scedosporium boydii), and Lomentospora prolificans were treated by intraperitoneal administration of olorofim (15 mg/kg of body weight every 8 h for 9 days). The efficacy of olorofim treatment was assessed by the survival rate at 10 days postinfection, levels of serum (1-3)-ß-d-glucan (BG), histopathology, and fungal burdens of kidneys 3 days postinfection. Olorofim therapy significantly improved survival compared to that of the untreated controls; 80%, 100%, and 100% of treated mice survived infection by Scedosporium apiospermum, Pseudallescheria boydii, and Lomentospora prolificans, respectively, while less than 20% of the control mice (phosphate-buffered saline [PBS] treated) survived at 10 days postinfection. In the olorofim-treated neutropenic CD-1 mice infected with any of the three species, serum BG levels were significantly suppressed and fungal DNA detected in the target organs was significantly lower than in controls. Furthermore, histopathology of kidneys revealed no or only a few lesions with hyphal elements in the olorofim-treated mice, while numerous fungal hyphae were present in control mice. These results indicate olorofim to be a promising therapeutic agent for systemic scedosporiosis/lomentosporiosis, devastating emerging fungal infections that are difficult to treat with currently available antifungals.

Efficacy of Olorofim (F901318) against Aspergillus fumigatus, A. nidulans, and A. tanneri in Murine Models of Profound Neutropenia and Chronic Granulomatous Disease.

The emergence of azole resistance in Aspergillus fumigatus as well as an increasing frequency of multiresistant cryptic Aspergillus spp. necessitates exploration of new classes of antifungals. Olorofim (formerly F901318) is a new fungicidal agent that prevents the growth of ascomycetous mold species via inhibition of de novo pyrimidine biosynthesis, a mechanism of action distinct from that of currently available antifungal drugs. We studied the in vivo efficacy of olorofim intraperitoneal therapy (15 mg/kg of body weight every 8 h for 9 days) against infection with A. fumigatus, A. nidulans, and A. tanneri in both neutropenic CD-1 mice and mice with chronic granulomatous disease (CGD) (gp91-/-phox mice). In the neutropenic mouse model, 80% to 88% of treated mice survived for 10 days, and in the CGD group, 63% to 88% of treated mice survived for 10 days, depending on the infecting species, while less than 10% of the mice in the control groups survived for 10 days. In the olorofim-treated groups, galactomannan levels were significantly suppressed, with lower organ fungal DNA burdens being seen for all three Aspergillus spp. Histopathological slides revealed a limited number of inflammatory foci with or without detectable fungal elements in the kidneys of neutropenic CD-1 mice and in the lungs of CGD mice. Furthermore, the efficacy of olorofim was unrelated to the triazole MICs of the infecting Aspergillus spp. These results show olorofim to be a promising therapeutic agent for invasive aspergillosis.

Inbred SJL mice recapitulate human resistance to Cryptococcus infection due to differential immune activation.

IMPORTANCE: Cryptococcosis studies often utilize the common C57BL/6J mouse model. Unfortunately, infection in these mice fails to replicate the basic course of human disease, particularly hampering immunological studies. This work demonstrates that SJL/J mice can recapitulate human infection better than other mouse strains. The immunological response to Cryptococcus infection in SJL/J mice was markedly different from C57BL/6J and much more productive in combating this infection. Characterization of infected mice demonstrated strain-specific genetic linkage and differential regulation of multiple important immune-relevant genes in response to Cryptococcus infection. While our results validate many of the previously identified immunological features of cryptococcosis, we also demonstrate limitations from previous mouse models as they may be less translatable to human disease. We concluded that SJL/J mice more faithfully recapitulate human cryptococcosis serving as an exciting new animal model for immunological and genetic studies.

Novel angular naphthopyrone formation by Arp1p dehydratase involved in Aspergillus fumigatus melanin biosynthesis.

Conidial pigment is an important virulence factor in Aspergillus fumigatus, a human fungal pathogen. The biosynthetic gene cluster for 1,8-dihydroxynaphthalene (DHN)-melanin in A. fumigatus consists of six genes, alb1, ayg1, arp1, arp2, abr1 and abr2. In contrast to black DHN-melanin fungi such as Magnaporthe grisea, the polyketide synthase Alb1p in A. fumigatus produces naphthopyrone YWA1 instead of 1,3,6,8-THN (T4HN) and YWA1 is converted to T4HN by Ayg1p. The yeast transformant expressing Alb1p and Arp1p dehydratase produced an unknown compound which was identified to be a novel angular naphthopyrone named YWA3 formed from YWA1. In addition, the amount of YWA3 produced was much more than that of YWA2 formed by non-enzymatic dehydration from YWA1. To further analyse the reaction in vitro, Arp1p was overexpressed in E. coli and purified. Kinetic analysis revealed Km value of Arp1p for YWA1 to be 41 µM which is comparable with that of Ayg1p for YWA1 in conversion to T4HN. The complex structure modelling well explained the mechanism of YWA3 generation by the dehydration of angular YWA1 by Arp1p. These results indicated the possibility that polymerization of angular naphthopyrone YWA3 but not YWA2 could be involved in the characteristic bluish-green conidial pigmentation of A. fumigatus.

Cryptococcus neoformans STE12alpha regulates virulence but is not essential for mating.

The Cryptococcus neoformans STE12alpha gene, a homologue of Saccharomyces cerevisiae STE12, exists only in mating type (MAT)alpha cells. In S. cerevisiae, STE12 was required for mating and filament formation. In C. neoformans, haploid fruiting on filament agar required STE12alpha. The ability to form hyphae, however, was not affected by deletion of STE12alpha when convergently growing MATa strains were present. Furthermore, ste12alpha disruptants were fertile when mated with MATa strains, albeit with reduced mating frequency. Most importantly, the virulence of a ste12alpha disruptant of serotype D strain was significantly reduced in a mouse model. When the ste12alpha locus was reconstituted with the wild-type allele by cotransformation, virulence was restored. Histopathological analysis demonstrated a reduction in capsular size of yeast cells, less severe cystic lesions, and stronger immune responses in meninges of mice infected with ste12alpha cells than those of mice infected with STE12alpha cells. Using reporter gene constructs, we found that STE12alpha controls the expression of several phenotypes known to be involved in virulence, such as capsule and melanin production. These results demonstrate a clear molecular link between mating type and virulence in C. neoformans.

Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans.

To assess the relationship between melanin production by Cryptococcus neoformans and virulence on a molecular basis, we asked: (a) is CNLAC1, the laccase structural gene of C. neoformans, expressed in vivo?; (b) can mouse virulence be restored to cnlac1 (Mel-) mutants by complementation with CNLAC1?; and (c) will targeted gene deletion of CNLAC1 decrease virulence for mice? Melanin is produced when cryptococcal laccase catalyzes the oxidation of certain aromatic compounds, including L-dopa, to quinones, which then polymerize to melanin. To assess CNLAC1 transcription, RNA was extracted from C. neoformans in cerebrospinal fluid of infected rabbits. Reverse transcriptase-polymerase chain reaction detected CNLAC1 transcript, indicating that laccase may be produced in the infected host. To assess the effect of CNLAC1 deletion on virulence, a Mel- mutant (10S) was obtained by disruption of the 5' end of the gene. After multiple backcrosses with a parental strain to remove unintended genetic defects introduced by the transformation process, a Mel- progeny was tested and found to be much less virulent for mice than a Mel+ progeny. Another Mel- strain (mel2), obtained from J.C. Edman (University of California at San Francisco, CA), produced CNLAC1 transcript but no detectable melanin. Characterization of this mutant revealed a base substitution in CNLAC1 that changed a histidine to tyrosine in a putative copper-binding site. When this base change was introduced into CNLAC1 by site-directed mutagenesis, it no longer transformed mel2 to Mel+, indicating the importance of this histidine in laccase activity. Complementation of a mel2-derived mutant with CNLAC1 restored the Mel+ phenotype and increased virulence. These results support the concept that the CNLAC1 gene product has a role in virulence.

Heteroresistance of Cryptococcus gattii to fluconazole.

We analyzed 71 clinical and environmental Cryptococcus gattii strains that had been isolated before or after the advent of azole antifungals to determine their level of heteroresistance to fluconazole (LHF). All strains of C. gattii manifested heteroresistance, with LHFs that ranged between 4 microg/ml and 32 microg/ml. A considerably higher proportion of the C. gattii strains (86%) than Cryptococcus neoformans strains (46%) exhibited LHFs that were > or =16 microg/ml. No significant correlation was observed between the molecular type or serotypes of strains and their respective LHF. The strains which expressed a higher LHF were also more resistant to xenobiotics than the strains with a low LHF, and the level of resistance to xenobiotics was significantly higher than that reported for C. neoformans. The heteroresistant subpopulation, whose level of drug resistance had been raised in a stepwise manner to 64 microg/ml, reverted to the original LHF upon daily transfers in drug-free medium. Importantly, the strains with high LHFs were significantly more virulent than those with low LHFs. Since all the clinical isolates that had not been exposed to azole drugs as well as the environmental strains manifested heteroresistance to fluconazole, heteroresistance of C. gattii to azoles is an intrinsic mechanism as in C. neoformans and is associated with the strain's virulence.

Death by edible mushroom: first report of Volvariella volvacea as an etiologic agent of invasive disease in a patient following double umbilical cord blood transplantation.

We describe a case of invasive fungal infection caused by Volvariella volvacea following double umbilical cord blood transplantation (UCBT). Although infections caused by several mushroom species have been documented, we believe this to be the first published report of invasive infection with Volvariella volvacea, an edible mushroom belonging to Agaricales.

Neosartorya udagawae (Aspergillus udagawae), an emerging agent of aspergillosis: how different is it from Aspergillus fumigatus?

A recent report on several cases of invasive aspergillosis caused by Neosartorya udagawae suggested distinctive patterns of disease progression between N. udagawae and Aspergillus fumigatus. This prompted us to characterize N. udagawae in comparison to A. fumigatus. Our findings showed that both species exist in two mating types at similar ratios and produce gliotoxin. However, the thermotolerance of the two species differs: while A. fumigatus is able to grow at 55 degrees C but not at 10 degrees C, N. udagawae is able to grow at 10 degrees C but fails to grow at >42 degrees C. Furthermore, compared to A. fumigatus, the conidia of N. udagawae require longer incubation periods to germinate at 37 degrees C and are more susceptible to neutrophil attack as well as hydrogen peroxide; N. udagawae is also less virulent in gp91(phox-/-) mice. These findings suggest that growth and susceptibility to the host response might account for the reduced virulence of N. udagawae and the subtle distinction in the progression of the disease caused by the two species.

Sexual stage of Histoplasma capsulatum.

Twelve primary subcultures of Histoplasma capsulatum, paired in all possible combinations on agar containing yeast extract and Alphacel, produced fertile cleistothecia, resembling those of Ajellomyces dermatitidis (Blastomyces dermatitidis).

The presence of capsule in Cryptococcus neoformans influences the gene expression profile in dendritic cells during interaction with the fungus.

The aim of this investigation was to study the effect of polysaccharide capsule on the gene expression in dendritic cells (DC) during their interaction with Cryptococcus neoformans. To this end, we used an encapsulated virulent strain of C. neoformans and a cap59 gene-disrupted acapsular avirulent strain derived from the same genetic background. DC were exposed to encapsulated and acapsular C. neoformans strains for 4 h and 18 h, and their transcriptional profiles were analyzed using the Affymetrix mouse gene chip U74Av2. A large number of DC genes were up-regulated after treatment with the acapsular strain. In particular, we observed the up-regulation of the genes involved in DC maturation, such as cell surface receptors, cytokines, and chemokines (interleukin-12 [IL-12], IL-2, IL-1alpha, IL-1beta, IL-6, IL-10, tumor necrosis factor alpha, CCR7, CCL17, CCL22, CCL3, CCL4, CCL7, and CXCL10), membrane proteins, and the genes involved in antigen processing and presentation as well as cell cycle or apoptosis. The chemokine gene expression data were confirmed by real-time reverse transcription-PCR, while the expression of cytokine genes was correlated with their secretion. A completely different pattern of gene expression was observed for DC treated with an encapsulated strain of C. neoformans. In particular, no significant induction was observed in the expression of the genes mentioned above. Moreover, a number of genes, such as those coding for chemokines, were down-regulated. These results suggest that the polysaccharide capsule shrouding the cell wall of C. neoformans plays a fundamental role in inducing DC response, highlighting the molecular basis of the true nature of immune silencing exerted by capsular material.

Emerging Aspergillus Species Almost Exclusively Associated With Primary Immunodeficiencies.

Invasive aspergillosis (IA) is the most serious mold infection encountered in patients with iatrogenic immunosuppression. IA is also a major cause of mortality and morbidity in individuals with primary immunodeficiency (PID). Although Aspergillus fumigatus is the most common etiologic agent of IA reported in PID patients, followed by A. nidulans, multiple poorly recognized Aspergillus species such as A. udagawae, A. quadrilineatus, A. pseudoviridinutans, A. tanneri, A. subramanianii, and A. fumisynnematus have been reported almost exclusively from patients with inborn defects in host antifungal defense pathways. Infection in PID patients exhibits patterns of disease progression distinct from those in iatrogenic immunosuppression. Specifically, the disease can be extrapulmonary and chronic with a tendency to disseminate in a contiguous manner across anatomical planes. It is also more refractory to standard antifungal therapy. This synopsis summarizes our understanding of emerging rare Aspergillus species that primarily affect patients with PIDs but not those with acquired immunodeficiencies.

Virulence of catalase-deficient aspergillus nidulans in p47(phox)-/- mice. Implications for fungal pathogenicity and host defense in chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare genetic disorder in which phagocytes fail to produce superoxide because of defects in one of several components of the NADPH oxidase complex. As a result, patients develop recurrent life-threatening bacterial and fungal infections. The organisms to which CGD patients are most susceptible produce catalase, regarded as an important factor for microbial pathogenicity in CGD. To test the role of pathogen-derived catalase in CGD directly, we have generated isogenic strains of Aspergillus nidulans in which one or both of the catalase genes (catA and catB), have been deleted. We hypothesized that catalase negative mutants would be less virulent than the wild-type strain in experimental animal models. CGD mice were produced by disruption of the p47(phox) gene which encodes the 47-kD subunit of the NADPH oxidase. Wild-type A. nidulans inoculated intranasally caused fatal infection in CGD mice, but did not cause disease in wild-type littermates. Surprisingly, wild-type A. nidulans and the catA, catB, and catA/catB mutants were equally virulent in CGD mice. Histopathological studies of fatally infected CGD mice showed widely distributed lesions in the lungs regardless of the presence or absence of the catA and catB genes. Similar to the CGD model, catalase-deficient A. nidulans was highly virulent in cortisone-treated BALB/c mice. Taken together, these results indicate that catalases do not play a significant role in pathogenicity of A. nidulans in p47(phox)-/- mice, and therefore raise doubt about the central role of catalases as a fungal virulence factor in CGD.

Isolation of the URA5 gene from Cryptococcus neoformans var. neoformans and its use as a selective marker for transformation.

A cDNA encoding Cryptococcus neoformans orotidine monophosphate pyrophosphorylase (OMPPase) has been isolated by complementation of the cognate Escherichia coli pyrE mutant. The cDNA was used as a probe to isolate a genomic DNA fragment encoding the OMPPase gene (URA5). By using electroporation for the introduction of plasmid DNA containing the URA5 gene, C. neoformans ura5 mutants could be transformed at low efficiency. Ura+ transformants obtained with supercoiled plasmids containing the URA5 gene showed marked mitotic instability and contained extrachromosomal URA5 sequences, suggesting limited ability to replicate within C. neoformans. Transformants obtained with linear DNA were of two classes: stable transformants with integrated URA5 sequences, and unstable transformants with extrachromosomal URA5 sequences.

Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence.

Capsule formation plays a significant role in the pathogenicity of Cryptococcus neoformans. To study the molecular basis of capsule synthesis, the capsule-deficient phenotype of a mutant strain was complemented by transformation. A plasmid rescued from the resulting Cap+ transformant complemented a cap59 mutation which was mapped previously by classical recombination analysis. Gene deletion by homologous integration resulted in an acapsular phenotype, indicating that we have identified the CAP59 gene. The CAP59 gene was assigned to chromosome I by Southern blot analysis of contour-clamped homogeneous electric field gel electrophoresis-resolved chromosomes of C. neoformans var. neoformans. Sequence comparison of genomic and cDNA clones indicated the presence of six introns. CAP59 encoded a 1.9-kb transcript and a deduced protein of 458 amino acids. Analysis of the nucleotide sequence revealed little similarity to existing sequences in the data bank. When the capsule-deficient phenotype was complemented, the originally avirulent C. neoformans strain became virulent for mice. In addition, the acapsular strain created by gene deletion of CAP59 lost its virulence. This work demonstrates the molecular basis for capsule-related virulence and that the CAP59 gene is required for capsule formation.

Doppler color flow mapping of simulated in vitro regurgitant jets: evaluation of the effects of orifice size and hemodynamic variables.

The spatial distribution of simulated regurgitant jets imaged by Doppler color flow mapping was evaluated under constant flow and pulsatile flow conditions. Jets were simulated through latex tubings of 3.2, 4.8, 6.35 and 7.9 mm by varying flow rates from 137 to 1,260 cc/min. Color jet area was linearly related to flow rate at each orifice (r = 0.96, SEE = 3.4; r = 0.99, SEE = 1.6; r = 0.97, SEE = 2.3; r = 0.97, SEE = 3.2, respectively), but significantly higher flow rates were required to maintain the same maximal spatial distribution of the jet at the larger regurgitant orifices. Constant flow jets were also simulated through needle orifices of 0.2, 0.5 and 1 mm, with a known total volume (5 cc) injected at varying flow rates and with differing absolute volumes injected at the same flow rate (0.2, 1.0 and 2.0 cc/s, respectively). Again, maximal color jet area was linearly related to flow rate at each orifice (r = 0.97, SEE = 2.3; r = 0.97, SEE = 2.4; r = 0.92, SEE = 3.9, respectively), but was not related to the absolute volume of regurgitation. Color encoding of regurgitant jets on Doppler color flow maps was demonstrated to be highly dependent on velocity and, hence, driving pressure, such that color encoding was obtained from a constant flow jet injected at a velocity of 4 m/s through an orifice of 0.04 mm diameter with flow rates as low as 0.008 cc/s. Mitral regurgitant jets were also simulated in a physiologic in vitro pulsatile flow model through three prosthetic valves with known regurgitant orifice sizes (0.2, 0.6 and 2.0 mm2). For each regurgitant orifice size, color jet area at each was linearly related to a regurgitant pressure drop (r = 0.98, SEE = 0.15; r = 0.97, SEE = 0.20; r = 0.97, SEE = 0.23, respectively), regurgitant stroke volume (r = 0.77, SEE = 0.55; r = 0.94, SEE = 0.30; r = 0.91, SEE = 0.41, respectively) and peak regurgitant flow rate (r = 0.98, SEE = 0.16; r = 0.97, SEE = 0.21; r = 0.93, SEE = 0.37, respectively), but the spatial distribution of the regurgitant jets was most highly dependent on the regurgitant pressure drop. Jet kinetic energy calculated from the summation of the individual pixel intensities integrated over the jet area was closely related to driving pressure (r = 0.84), but integration of the power mode area times pixel intensities provided the best estimation of regurgitant stroke volume (r = 0.80).(ABSTRACT TRUNCATED AT 400 WORDS)

Digital subtraction angiography in patients with transposition of the great arteries after surgical repair.

Digital subtraction angiography was used for postoperative evaluation of seven patients who underwent the Senning procedure for repair of d-transposition of the great arteries. Their ages ranged from 2.5 to 3 years. The patients were premedicated with methohexital (25 mg/kg rectally), and 0.3 to 0.4 ml/kg of diatrizoate was injected into a peripheral vein through a plastic needle. Images were obtained on a Technicare DR-960 or Diasonics DA 100 digital angiographic unit at four frames per second using 256 X 256 matrix and a 6 inch (15.24 cm) field size. In all patients, the venous systems, cardiac chambers and great arteries were well visualized. Two patients had obstruction of the superior vena cava with a dilated azygos vein draining into the inferior vena cava. One patient had severe obstruction of the left pulmonary artery. Digital subtraction angiography is safe and easy to perform and appears to be a valuable alternative method for evaluating patients after surgical repair of transposition of the great arteries. The small amount of contrast material required and the low radiation dose make it attractive for use in children.

Factors influencing the structure and shape of stenotic and regurgitant jets: an in vitro investigation using Doppler color flow mapping and optical flow visualization.

To evaluate factors influencing the structure and shape of stenotic and regurgitant jets, Doppler color flow mapping and optical flow visualization studies were performed with use of a syringe model with a constant rate of ejection to simulate jets of valvular regurgitation and a pulsatile flow model of the right heart chambers to simulate jets of mild, moderate and severe valvular pulmonary stenosis. Ink-(0 to 40%) glycerol-water jets (viscosity 1 to 3.5 centiPoise) were produced by injecting the fluid at a constant rate into a 10 gallon rectangular reservoir of the same still fluid through 1.4 and 3.4 mm needles. The Doppler color flow scanners imaged the laminar jet length within 3 mm of actual jet length (2 to 6 cm) and the jet width within 2 to 3 mm of the actual jet width. Jet flows with Reynolds numbers ranging from 230 to 1,200 injected into still fluid yielded jet length/width ratios that decreased with increasing Reynolds numbers and leveled off to a length/width ratio of 5-6:1 at a Reynolds number near 600. When the fluid reservoir was swirled to better mimic the effect of flow entering the same cardiac chamber from a second source, the jets showed diminution of the jet length/width ratio and a clearly defined zone of turbulence. Studies of the pulsatile flow model were performed at cardiac outputs of 1 to 6 liters/min for the normal and each stenotic valve. Mild stenosis had an orifice area of 2.8 cm2, moderate stenosis an area of 1.0 cm2 and severe stenosis an area of 0.5 cm2. Laminar jet length represented the length of the total jet, which had a symmetric width and was measured from the valve opening to a region where the jet exhibited a spray effect. Laminar jet lengths (0.2 to 1.1 cm) were imaged by Doppler color flow mapping and optical visualization only in the moderate and severely stenotic valves and only at flows less than or equal to 3 liters/min (mean Reynolds numbers less than or equal to 3,470). Beyond this flow rate the jets exhibited a spray effect. Laminar jet length/width ratio approached unity with an increased amount of valvular stenosis and higher flow volumes (cardiac output). Proximal aliasing was present in each valve studied. the length of aliasing (0 to 3.2 cm) proximal to the valve was longer with increased flow rates and increased amounts of stenosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Real-time Doppler color flow mapping for detection of patent ductus arteriosus.

In this study, ultrasound Doppler color flow mapping systems were utilized to examine flow in the pulmonary artery in 31 premature and term infants (aged 4 hours to 9 months) with patent ductus arteriosus accompanying respiratory distress syndrome, as an isolated lesion, or with patent ductus in association with other cyanotic or acyanotic congenital heart disorders. The flow mapping patterns were compared with those of a control population of 15 infants who did not have patent ductus arteriosus. In unconstricted ductus arteriosus, the flow from the aorta into the pulmonary artery was detected in late systole and early diastole and was distributed along the superior leftward lateral wall of the main pulmonary artery from the origin of the left pulmonary artery back in a proximal direction toward the pulmonary valve. In constricted patent ductus arteriosus, or especially in a ductus in association with cyanotic heart disease, the position of the ductal shunt in the pulmonary artery was more variable, often directed centrally or medially. Waveform spectral Doppler sampling could be performed in specific positions guided by the Doppler flow map to verify the phasic characteristics of the ductal shunt on spectral and audio outputs. Shunts through a very small patent ductus arteriosus were routinely detected in this group of infants, and right to left ductal shunts could also be verified by the Doppler flow mapping technique. This study suggests substantial promise for real-time two-dimensional Doppler echocardiographic flow mapping for evaluation of patent ductus arteriosus in infants.