Scopulariopsis Species Research Articles

Mycoflora of Crude Oil Polluted Soils of Ukwa West of Abia State Nigeria

Aim: Bioremediation of soil play a critical role in removing pollutants from crude oil polluted soil. To investigate the mycoflora of crude oil polluted area of Ukwa West Local Government in the present Abia State.
 Methodology: Portions of the soil samples collected from three (3) locations were plated out separately on Yeast Starch Agar, Starch Agar, and Cellulose Agar using soil plate methods. The culture plates were examined after 5-7 and 14 days for the presence of fungi. Pure cultures of the isolates were obtained by subculturing and the physico-chemical properties of the soil samples were determined using standard methods.
 Results: Twenty species (20) of fungi were isolated from the crude oil polluted soil sample and represented by their respective genera. Eight (8) species of Aspergillus representing 40%, two (2) species of Thermomyces representing 10% each, and one (1) species of Penicillium, Cladosporium, Cunninghamella, Curvularia, Trichoderma, Scopulariopsis, Sporotrichum and Basipetospora representing 5% each. Aspergillus species were predominant which include among others A. fumigatus, A. niger, A. parasiticus, A. oryzae, A. terreus. The physico-chemical properties of the soil sample were found to be varied and have affected the distribution and population of fungi. The pH values ranged from 4.81- 5.58 as compared to the control ranging from 5.72- 6.50 for soil samples A, B and C. The moisture content values ranged between 26.46-29.59% as compared to the control ranging from 29.41-32.51%. The soil was found to be high in organic matter content with values of 70.3-82.7% as compared to the control which was 94% for each of the locations.
 Conclusion: Crude oil polluted soils of Ukwa West of Abia State, Nigeria is rich in fungal biodiversity with the soil’s samples having different physico-chemical properties.

Two new Microascus species with spinous conidia isolated from pig farm soils in China

Abstract Two scopulariopsis-like fungi were isolated when surveying fungi in pig farm soils in China. Sexual structures of these fungi were not observed and their conidia had spinous walls. Phylogenetic analysis based on nucleotide sequences of the internal transcribed spacer 1 and 2 and intervening 5.8S ribosomal RNA gene (ITS), large subunit ribosomal RNA gene (LSU), beta-tubulin (tub2) and translation elongation factor 1-alpha (tef1) gene showed that they were new members of the genus Microascus, and the name M. aculeatus and M. spinosporus were introduced. Genetically the two new species clustered in a well-supported clade close to M. longicollis, but differed in producing relatively long branches. Morphologically M. aculeatus could be distinguished by its conidia with sparse but long spines; M. spinosporus resembled three species of the genus Scopulariopsis, S. asperula, S. brevicaulis and S. flava, but was different in the color of colony and conidia, and the form of conidiophores.

Invasive Scopulariopsis alboflavescens infection in patient with acute myeloid leukemia

Scopulariopsis alboflavescens is a soil saprophyte that is widely distributed in nature. Recently, there have been increasing number of reports of invasive infections with Scopulariopsis species in immunocompromised patients. In this report, we described an adult woman with acute myeloid leukemia and who developed S. alboflavescens pneumonia. Liposomal amphotericin B and voriconazole combination therapy was unsuccessful and the patient died because of pneumonia. Scopulariopsis is highly resistant to available antifungal agents and almost invariably fatal. This case report should alert clinicians to the importance of listing Scopulariopsis as a pathogenic fungus in immunocompromised patients.

Necrotizing Microascus tracheobronchitis in a bilateral lung transplant recipient

Invasive fungal infections are a major cause of mortality among solid organ transplant recipients. Scopulariopsis species and their teleomorph Microascus are molds found in soil and decaying organic matter. We report here the case of a woman who underwent bilateral lung transplantation for severe emphysema. On day 25 after transplantation, endobronchial green-black lesions were detected during routine endoscopy. Endobronchial swabs, biopsies, and bronchoalveolar lavage samples were positive for Microascus cirrosus. This fungal infection developed despite voriconazole given for previous persistent invasive aspergillosis. Treatment consisted of a combination of antifungal medication (voriconazole, terbinafine, amphotericin B, and caspofungin) and endoscopic resection of necrosed bronchial mucosa. A favorable clinical outcome was achieved after 7weeks of treatment. Seven cases of Scopulariopsis/Microascus infection have been previously described in solid organ transplant recipients. Only two survived after treatment with an antifungal combination therapy including echinocandins, posaconazole, and terbinafine. In immunocompromised patients, infection by Microascus species is a rare but life-threatening event because of innate resistance to most common antifungal drugs. Our patient was successfully cured by combined therapy including intravenous voriconazole and caspofungin, oral terbinafine, and inhaled voriconazole and amphotericin B administered for 7weeks in association with iterative endoscopic debridement to reduce fungal inoculum.

A new species of Scopulariopsis and its synergistic effect on pathogenicity of Verticillium dahliae on cotton plants

A new species, Scopulariopsis gossypii, was found to be present in the vascular bundles of cotton plants (Gossypium hirsutum) infected by Verticillium dahliae which is an economically important pathogen in Hangzhou, China. The fungus was only present in the diseased plants, but it never became isolated from the healthy plants. Scanning electron microscopy showed that the fungus was present in parenchyma cells enclosing vessels in dark brown vascular tissues of stems, and produced asexual conidia within the tissues. Phylogenetic analysis of combined nuclear ribosomal D1/D2 region of the 28S rDNA as well as translation elongation factor 1-alpha (TEF1) and beta-tubulin (TUB) gene showed that S. gossypii represents an undescribed species of Scopulariopsis, Microascaceae. In this study, characteristics of sexual and asexual stages of the fungus were described, illustrated and compared with similar taxa. In addition, the molecular and morphological analyses indicated that S. gossypii was a distinct species of Scopulariopsis. The pathogenicity tests proved by inoculation of wounded roots confirmed that S. gossypii was an opportunistic pathogen causing leaf interveinal chlorosis and vascular browning of cotton plants. However S. gossypii did not infect host with undamaged roots. Moreover, coinoculation with S. gossypii and V. dahliae significantly increased disease severity.

Scopulariopsis and scopulariopsis-like species from indoor environments.

Scopulariopsis-like species are often reported from the indoor environment, as well as from clinical samples. The lack of type isolates and thorough phylogenetic studies in the Microascaceae hampered the correct identification of these isolates. Based on recent phylogenetic studies, which resulted in multiple name changes, the aim is to molecularly identify the Scopulariopsis and scopulariopsis-like species which occur in the indoor environment and give an overview of the current species in these genera and their habitats. Strains from the CBS culture collection were supplemented with almost 80 indoor strains of which the internal transcribed spacer 1 and 2 and intervening 5.8S nrDNA (ITS), beta-tubulin (tub2) and translation elongation factor 1-alpha (tef1) gene regions were sequenced for phylogenetic inference. The multi-gene phylogenies recognise 33 Microascus species and 12 Scopulariopsis species and showed that the recently established genus Fuscoannellis, typified by Scopulariopsis carbonaria, should be synonymized with the genus Yunnania. Seven new Microascus species, four new Scopulariopsis species, and one new Yunnania species, are described, and a new name in Microascus and two new name combinations (one in Microascus, and one in Yunnania) are proposed. In the indoor environment 14 Microascus species and three Scopulariopsis species were found. Scopulariopsis brevicaulis (22 indoor isolates) and Microascus melanosporus (19 indoor isolates) are the most common indoor species, in number of isolates, followed by M. paisii (8 indoor isolates) and S. candida (7 indoor isolates). A genus phylogeny based on the ITS, tef1 and the large subunit 28S nrDNA (LSU) of the type or representative isolates of all here recognised species is provided depicting all species habitats. No correlation between phylogenetic relationship and habitat preference could be observed. Ten species which are found indoor are also found in relation with human-derived samples. A table showing recent name changes and a key to common species of Scopulariopsis and scopulariopsis-like genera found indoors is included.

Incidence and biodiversity of yeasts, dermatophytes and non-dermatophytes in superficial skin infections in Assiut, Egypt

The aim was to identify the incidence of the causal agents from dermatophytes, non-dermatophytes and yeasts in Assiut Governorate employing, beside the morphological and physiological techniques, the genotypic ones. Samples from infected nails, skin and hair were taken from 125 patients. Patients who presented with onychomycosis, tinea capitis, tinea corporis, tinea cruris and tinea pedis during the period from February 2012 to October 2015 were clinically examined and diagnosed by dermatologists and were guided to Assiut University Mycological Centre for direct microscopic examination, culturing and identification. Onychomycosis was the most common infecting (64.8% of the cases) followed by tinea capitis (17.6%). Direct microscopic preparations showed only 45 positive cases, while 96 cases showed positive cultures. Infections were more frequent in females than males. Fifty-one fungal species and 1 variety were obtained. Yeasts were the main agents being cultured from 46.02% of total cases. Non-dermatophytes were the second etiologic agents. Aspergillus was responsible for infecting 19.47% of total cases and dermatophytes appeared in only 15.93% of the cases. Yeasts were the main causal agents followed by non-dermatophytic fungi (mainly species of Aspergillus, then Alternaria, Scopulariopsis and Fusarium). Both direct microscopic preparations and culturing are recommended for mycological evaluation of clinical specimens. Sequence analysis of ITS region is recommended for yeast identification.

Epidemiology of invasive respiratory disease caused by emerging non‐Aspergillus molds in lung transplant recipients

Our aim was to assess the impact of positive cultures for non-Aspergillus molds on the risk of progression to invasive fungal infection (IFI), and the effect of prophylactic nebulized liposomal amphotericin B (n-LAB) on these pathogens. This was an observational study (2003-2013) including lung transplant recipients (LTR) receiving lifetime n-LAB prophylaxis, in whom non-Aspergillus molds were isolated on respiratory culture before and after transplantation (minimum 1-year follow-up). We studied 412 patients, with a mean postoperative follow-up of 2.56 years (interquartile range 1.01-4.65). Pre- and post-transplantation respiratory samples were frequently positive for non-Aspergillus molds (11.9% and 16.9% of LTR respectively). Post transplantation, 10 (2.42%) patients developed non-Aspergillus mold infection (4 Scedosporium species, 4 Purpureocillium species, 1 Penicillium species, and 1 Scopulariopsis species); 5 (1.21%) had IFI, with 60% IFI-related mortality. Non-Aspergillus molds with intrinsic amphotericin B (AB) resistance were more commonly isolated in bronchoscopy samples than AB-variably sensitive or AB-sensitive molds (54.5% vs. 25%, P = 0.04) and were associated with a higher risk of infection (56.3% vs. 1.3%%, P < 0.01). In LTR undergoing n-LAB prophylaxis, pre- and post-transplantation isolation of non-Aspergillus molds is frequent, but IFI incidence (1.21%) is low. Purpureocillium is an emerging mold. AB-resistant non-Aspergillus species were found more often in bronchoscopy samples and were associated with a higher risk of infection.

Virulence and Resistance to Antifungal Therapies of Scopulariopsis Species.

Scopulariopsisis an emerging opportunistic fungus characterized by its high resistance to antifungal therapies. We have developed a murine model of disseminated infection in immunosuppressed animals by intravenous inoculation ofScopulariopsis brevicaulisandScopulariopsis brumptii, the most clinically relevant species, in order to evaluate their virulence and their responses to conventional antifungal treatments. Survival and tissue burden studies showed thatS. brumptiiwas more virulent thanS. brevicaulis The three drugs tested, liposomal amphotericin B, posaconazole, and voriconazole, prolonged the survival of mice infected withS. brumptii, but none showed efficacy againstS. brevicaulis The different therapies were only able to modestly reduce the fungal burden of infected tissue; however, in general, despite the high serum levels reached, they showed poor efficacy in the treatment of the infection. Unfortunately, the most effective therapy forScopulariopsisinfections remains unresolved.

Aerosol distribution during open suctioning and long-term surveillance of air quality in a respiratory care center within a medical center.

The investigation of hospital air quality has been conducted in wards, ICUs, operating theaters, and public areas. Few studies have assessed air quality in respiratory care centers (RCCs), especially in mechanically ventilated patients with open suctioning. The RCC air quality indices (temperature, relative humidity, levels of CO2, total volatile organic compounds, particulate matter [PM], bacteria, and fungi) were monitored over 1 y. The air around the patient's head was sampled during open suctioning to examine the probability of bioaerosol exposure affecting health-care workers. This investigation found that the levels of indoor air pollutants (CO2, PM, bacteria, and fungi) were below the indoor air quality standard set by the Taiwan Environmental Protection Agency. Meanwhile, the levels of total volatile organic compounds sometimes exceeded the indoor air quality standard, particularly in August. The identified bacterial genera included Micrococcus species, Corynebacterium species, and Staphylococcus species, and the predominant fungal genera included yeast, Aspergillus species, Scopulariopsis species, and Trichoderma species. Additionally, airborne PM2.5, PM1, and bacteria were clearly raised during open suctioning in mechanically ventilated patients. This phenomenon demonstrated that open suctioning may increase the bacterial exposure risk of health-care workers. RCC air quality deserves long-term monitoring and evaluation. Health-care workers must implement self-protection strategies during open suctioning to ensure their occupational health and safety in health-care settings.

Prevalence of dermatophytes and other superficial fungal organisms in asymptomatic guinea pigs in Southern Italy

Guinea pigs have been indicated as a potential source of zoophilic dermatophytes that cause human dermatomycosis. The purpose of this study was to evaluate the prevalence of dermatophytes as well as saprophytic fungi in asymptomatic pet guinea pigs in Southern Italy. Two-hundred pet guinea pigs were enrolled from both private veterinary clinics and pet shops in the Campania region, Italy, from August 2012 to September 2013. Samples were collected using the MacKenzie's toothbrush technique. The plates were incubated for four weeks at 25°C and identification of the fungal colonies was based on both macroscopic and microscopic characteristics. Two pathogenic dermatophytes were isolated in 9 (4·5%) of 200 guinea pigs; Epidermophyton species in 2 (1%) and Scopulariopsis species in 7 (3·5%). Saprophytic dermatophytes were isolated from 151 (75·5%) animals enrolled. No fungal growth was observed in 40 (20%) guinea pigs. The results of this study indicate a low prevalence of pathogenic dermatophytes in pet guinea pigs in Southern Italy but the presence of Epidermophyton and Scopulariopsis species in asymptomatic pet guinea pigs.

Identification of Scopulariopsis Species by Partial 28S rRNA Gene Sequence Analysis

The genus Scopulariopsis contains over 30 species of mitosporic moulds, which although usually saprophytic may also act as opportunistic pathogens in humans. They have mainly been associated with onychomycosis, and only sporadically reported as a cause of deep tissue infections or systemic disease. Identification of Scopulariopsis species still largely relies on phenotype-based methods. There is a need for a molecular diagnostic approach, that would allow to reliably discriminate between different Scopulariopsis species. The aim of this study was to apply sequence analysis of partial 28S rRNA gene for species identification of Scopulariopsis clinical isolates. Although the method employed did reveal some genetic polymorphism among Scopulariopsis isolates tested, it was not enough for species delineation. For this to be achieved, other genetic loci, within and beyond the rDNA operon, need to be investigated.

A Case of Misidentification ofAspergillus versicolorComplex asScopulariopsisSpecies Isolated from a Homograft

A Case of Misidentification of Aspergillus versicolor Complex as Scopulariopsis Species Isolated from a Homograft Hee Jae Huh, Jang Ho Lee, Kyung Sun Park, Tae-Gook Jun, I-Seok Kang, Yae-Jean Kim, Chang-Seok Ki, Nam Yong Lee Departments of Laboratory Medicine & Genetics, Thoracic and Cardiovascular Surgery, and Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea

The Prevalence of Fungi in Soil of Qazvin, Iran

Backgrounds: Soil is the first habitat for pathogenic fungi and one of the important fungal reservoirs. The soil-borne fungi are major cause for different degrees of allergy or serious diseases in human beings. Objectives: This current study aimed to determine the identities and diversity of the fungi associated with allergy, subcutaneous and even systemic fungal infections in an extensive region of Qazvin district Iran during 2008. Materials and Methods: In this study, 150 soil samples were randomly collected from 25 locations of the city. The soil suspensions were prepared by sterile saline and then antibiotics of streptomycin and penicillin were added and a portion of supernatant was cultured on sabouraud’s dextrose agar culture medium containing chloramphenicol with and without cycloheximide and incubated at 35°C for 2-3 weeks. The fungal isolates were examined macroscopically and microscopically. Results: Plates were tested to be positive for saprophytic fungi. Out of a total of 1563 fungi isolates, 14 genera were diagnosed. Cladosporium species representing (29.69%) of isolates, followed by species of Aspergillus (22.52%), Penicillium (19.90%), Rhizopus (8.38%), Alternaria (6.59%), Mucor (3.77%), Chrysosporium (1.98%), Acremonium (1.60%), Fusarium (1.34%), Mycelia Sterilia (0.90%), Ulocladium (0.83%), Drechslera (0.58%) and Scopulariopsis (0.45%). Conclusions: The wide range of fungi, especially fungi associated with allergy, found in the soil of Qazvin indicated that this area was apt to propagate other medically important fungi and although none of the dimorphic ones were documented, there is always the possibility to find these diseases in the country and it is essential that physicians be aware of diseases produced by these fungi, especially in predisposed people.

InvasiveScopulariopsis brevicaulisinfection in an immunocompromised patient and review of prior cases caused byScopulariopsisandMicroascusspecies

Scopulariopsis species and their Microascus teleomorphs are cosmopolitan fungi that are uncommonly associated with invasive disease. This report describes a case of fatal disseminated Scopulariopsis brevicaulis disease in a patient with diffuse large B cell lymphoma who underwent high-dose chemotherapy followed by a matched unrelated donor stem cell transplant. This case is compared with 32 prior cases of proven invasive Scopulariopsis (Microascus) infections reported in the literature. A focus of this report is the diagnostic methods utilized which included histopathology and culture with both micromorphologic and genotypic procedures employed to confirm the species identification.

A taxonomic and ecological overview of cheese fungi

Cheese is made from milk by a succession of microbes (bacteria, yeasts and fungi) that determine the consistency and flavor of the cheese. Apart from the emblematic species, Penicillium camemberti and Penicillium roqueforti, cheese fungi are not well known. Here we present a taxonomic and phylogenetic overview of the most important filamentous cheese Ascomycota based on 133 isolates provided by the producers of cheese and cheese starter cultures and 97 isolates from culture collections. We checked the congruence of different gene genealogies to circumscribe cheese species and our results allow us to propose molecular targets for their identification. To study their phylogenetic affiliation, we used LSU rDNA and showed that cheese fungi are found in two classes, the Eurotiomycetes with Penicillium species (Eurotiales) and Sporendonema casei/Sphaerosporium equinum (Onygenales), and the Sordariomycetes with Scopulariopsis species (Microascales) and Fusarium domesticum (Hypocreales). Some of these fungi, such as, P. camemberti, F. domesticum, Scopulariopsis flava and S. casei, are only known from cheeses and are probably adapted to this particular habitat, which is extremely rich in protein and fat. Other cheese fungi are ubiquitous, such as, P. roqueforti, Scopulariopsis candida and Scopulariopsis fusca.

Rapid In-situ Hybridization for Dematiaceous Fungi Using a Broad-spectrum Oligonucleotide DNA Probe

Dematiaceous fungi are a diverse group of "darkly" pigmented fungi, which contain melanin in their cell walls and are commonly found in soil worldwide. Although morphology and histochemical stains may aid identification in tissue sections, these means for species identification are not specific. In-situ hybridization (ISH) for abundant fungal rRNA sequences may provide a means for detecting dematiaceous fungi. In this study, a 24-base synthetic biotin-labeled oligonucleotide probe targeting rRNA sequences of a variety of dematiaceous fungi was developed. This probe was tested on a cohort of 29 patients with culture-proven cases of dematiaceous fungal-associated rhinosinusitis (26 allergic fungal sinusitis, 2 fungal ball, and 1 acute invasive fungal sinusitis). Fungal cultures were positive for Alternaria species (10), Bipolaris species (5), Curvularia species (10), Cladosporium species (1), Scedosporium prolificans (1), Scopulariopsis species (1), and dematiaceous species, not otherwise specific (1). ISH showed positivity in fungal organisms in 24 of 29 specimens. ISH was negative in culture-proven examples of Rhizopus species, Aspergillus species, Fusarium species, Paecilomyces species, Histoplasmosis capsulatum, Candida species, and Blastomyces dermatitidis. ISH with a dematiaceous-specific fungal probe may be useful for differentiating dematiaceous fungi from other filamentous fungi in tissues, particularly those responsible for fungal rhinosinusitis.

Scopulariopsis brevicaulis abscess in a child treated for myeloblastic leukaemia

An 11-month-old boy from Reunion, France, was admitted for acute monoblastic leukaemia with skin, meningeal, and testicular leukaemic localisations. Initial white blood count was 200 cells per μL. Intensive chemotherapy led to severe pancytopenia around day 8. On day 12, a temperature of 38·6°C prompted antibiotic therapy with imipenem, cilastatin, and amikacin, which was completed on day 15 with vancomycin. On day 21, the child presented a recurrence of fever with cough and moderate polypnoea. Auscultation indicated a decreased vesicular murmur in the left lung fi eld, and liposomal amphotericine B (3 mg/kg per day) was started. A CT scan showed thoracic distension and an abscessed compressive lesion encompassing the left main stem bronchus (fi gure, panels A and B). For the next 7 days, the patient remained febrile without further radiographic improvement. Antifungal treatment was modifi ed accordingly to caspofungin (1·5 mg/kg per day, after fi rst dose of 2 mg/kg) and intravenous voriconazole (7 mg/kg per day). A fi bre-optic bronchoscopy done on day 29 showed a whitish lesion, resulting in complete obstruction of the left main stem bronchus (fi gure, panel C). Direct mycobacteriological examination was negative. Tests on the sampled culture identifi ed a Scopulariopsis brevicaulis infection. Haematological recovery occurred on day 35, with apyrexia. The patient was in complete remission for leukaemia. Progression of illness was marked by fi stulisation of the abscess in the bronchus, development of pneumomediastinum, and, subsequently, complete atelectasis of the left lung. Treatment with caspofungin and voriconazole was maintained daily and intravenously for 3 months. Then, oral voriconazole (150 mg twice daily) was given in addition to casponfungin (1·5 mg/kg three times a week) for 3 months while the patient received the last chemotherapy courses. Oral voriconazole was given alone daily for the next 6 months. The patient was alive at 18 month follow-up, with a complete atelectasis of the left lung, and without fungal or haematological relapses. Scopulariopsis species are fi lamentous fungi, which are common soil saprophytes. Onychomycosis and localised infections have been reported in immunocompetent patients. Although invasive infections are rare, most occur in immunocompromised hosts and are associated with high mortality. Most antifungals have limited in vitro activity against Scopulariopsis species. Confl icts of interest We declare that we have no confl icts of interest.

Scopulariopsis brevicaulis infection of prosthetic valve resembling aspergilloma on histopathology

Fungal prosthetic valve endocarditis is an infrequent but serious complication of valve replacement surgery. Approximately 75% of patients are affected with Candida species. Scopulariopsis species affecting prosthetic valve is rarely reported in the literature.

Impact of Production Systems on Swine Confinement Buildings Bioaerosols

Hog production has been substantially intensified in Eastern Canada. Hogs are now fattened in swine confinement buildings with controlled ventilation systems and high animal densities. Newly designed buildings are equipped with conventional manure handling and management systems, shallow or deep litter systems, or source separation systems to manage the large volumes of waste. However, the impacts of those alternative production systems on bioaerosol concentrations within the barns have never been evaluated. Bioaerosols were characterized in 18 modern swine confinement buildings, and the differences in bioaerosol composition in the three different production systems were evaluated. Total dust, endotoxins, culturable actinomycetes, fungi, and bacteria were collected with various apparatuses. The total DNA of the air samples was extracted, and quantitative polymerase chain reaction (PCR) was used to assess the total number of bacterial genomes, as a total (culturable and nonculturable) bacterial assessment. The measured total dust and endotoxin concentrations were not statistically different in the three studied production systems. In buildings with sawdust beds, actinomycetes and molds were found in higher concentrations than in the conventional barns. Aspergillus, Cladosporium, Penicillium, and Scopulariopsis species were identified in all the studied swine confinement buildings. A. flavus, A. terreus, and A. versicolor were abundantly present in the facilities with sawdust beds. Thermotolerant A. fumigatus and Mucor were usually found in all the buildings. The culturable bacteria concentrations were higher in the barns with litters than in the conventional buildings, while real-time PCR revealed nonstatistically different concentrations of total bacteria in all the studied swine confinement buildings. In terms of workers' respiratory health, barns equipped with a solid/liquid separation system may offer better air quality than conventional buildings or barns with sawdust beds. The impact of ventilation rates, air distribution, or building design still has to be explored.