Prototheca spp. : Interest in human and animal pathology and sources of contamination (environmental samples, food, and samples of animal origin) – Cultue; Molecular diagnosis (PCR) ; Molecular identification (PCR and sequencing)
The genus Prototheca includes unicellular, aerobic, achlorophilic, and ubiquitously distributed algae in the environment. Prototheca algae live predominantly as saprophytes in aqueous environments containing decomposing plant material. However, some Prototheca species can cause an infectious disease in humans and animals called protothecosis. Previously, the genus Prototheca was considered a rare pathogen, but recently, an increasing number of cases of infection by these algae have been reported in animals and humans, including opportunistic infection in immunosuppressed patients. In animals, prototecal bovine mastitis produces considerable economic losses for the dairy industry worldwide. While in humans, given the growing number of immunocompromised patients worldwide, the incidence of infection caused by these organisms is increasing. Therefore, the prevention and control of the presence of Prototheca spp. in the main sources of contagion it is increasingly important.
The genus Prototheca was first described by Krüger in 1894, as unicellular microorganisms, not pigmented, isolated from the resin of the trees, and then considered as yeast fungi, because in culture media they can produce colonies of similar appearance to the of the yeasts. Currently, Prototheca spp. are recognized as algae, and assigned to the class Trebouxiophyceae, family Chlorelaceae. They are spherical organisms, whose diameter varies from 3 to 30 μm in diameter and they reproduce asexually by releasing endospores. They are closely related to the green algae genus Chlorella, a genus that is currently believed to have evolved. However, the genus Prototheca is composed of colorless algae, chlorophyll, which have lost their photosynthetic capacity, and are saprophytic inhabitants in the environment, which under certain conditions, give rise to infections in humans and animals. The genus Prototheca constitutes the only genus of plants recognized as causal agents of human and animal diseases.
Currently, seven species are recognized within the genus Prototheca: P. wickerhamii, P. zopfii, P. blaschkeae, P. cutis, P. ulmea, P. stagnora, and P. mirajii. In addition, the species P. wickerhamii and P.zopfii have differentiated into two genotypes, gen 1 and gen 2. Of the seven species currently postulated, five (P. zopfii, P. wickerhamii, P. blaschkeae, P. cutis, and P. miyajii) are described as opportunistic pathogens of humans and animals. Prototheca species are widespread throughout the world, and can be isolated from various reservoirs, such as the environment, animals and food. The typical sources of Prototheca species are resin from trees, grass, fresh and salt water, sewage, land, animals (such as cattle, deer and dogs), stables, drinking fountains, excrement and digestive tract of men and animals, and food (such as butter, potato skin, fruit, and cow's milk and its derivatives). In addition, each species or strain of Prototheca has a different susceptibility to commercial chlorinating agents. As a result, chlorination is not uniformly effective in the elimination of potentially pathogenic Prototheca species from wastewater and domestic waste effluents, so that algae survive and return to the environment.
Human prototecosis is a cosmopolitan disease, although it is more frequent in tropical areas. Prototheca infection can occur in both, immunocompetent and immunosuppressed patients, although more severe and disseminated infections tend to occur in immunocompromised patients. The species that most commonly causes human protothecosis is P. wickerhamii, followed by P. zopfii. The pathogenesis of this infection is largely unknown. It is believed that Prototheca species can infect humans by contacting the skin with potential sources, such as water, soil, swimming pools or water tanks, farm animals, and work sites with permanent moisture. Also, it is thought that in many cases the organisms manage to enter through open wounds, traumatisms, surgical wounds, or even, through insect bites. It is admitted that protothecosis is not transmitted from person to person. Susceptibility to infection is often linked to some form of immunosuppression. Risk factors include: surgery, diabetes mellitus, treatment with immunosuppressants, kidney transplantation, HIV infection and AIDS, and chemotherapy or radiation therapy. The incubation period of the disease is not known, but it is thought that it may take weeks to months.
Clinically, protothecosis can manifest itself in three forms, skin lesions, olecranon bursitis, and disseminated or systemic infections. In addition, rare presentations such as protothecosis of the urinary tract, respiratory tract, intestine, colpitis, choroiditis, inguinal infections, and meningitis have been documented. The skin is the most frequently affected organ in the protothecal infection. Cutaneous lesions can occur in a variety of ways. At least half of the cases of protothecosis are simple skin infections, and most of these occur in individuals undergoing immunosuppressive therapy. In contrast, individuals who present with olecranon bursitis are not usually immunocompromised, but have a trauma, penetrating or not, in the affected elbow. Signs and symptoms appear gradually several weeks after the trauma and include mild induration of the pouch, accompanied by sensitivity, erythema, and production of varying amounts of serosanguineous fluid. Finally, the dissemination of Prototheca spp. occurs in severely immunosuppressed individuals. The most commonly affected organs in systemic protothecosis are the skin, subcutaneous tissue, intestine, peritoneum, blood and spleen. In most of the described cases of disseminated infection, the species involved was P. wickerhamii.
In general, the treatment of human protothecosis involves medical and surgical approaches, and failure of treatment is not uncommon. Algae of the genus Prototheca have a high resistance to antibacterial and antifungal antibiotics. Antifungals such as ketoconazole, itraconazole, fluconazole and amphotericin B are the most commonly used medications to date. Among them, amphotericin B shows the best activity against Prototheca spp.
Protothecosis in animals
In animals, protothecosis manifests mainly in cattle and dogs. In bovines, it usually causes mastitis, which is the most common protothecosis in animals. Bovine mastitis is caused by P. zopfii gen. 2 mainly, but also by P. blaschkeae and P. wickerhamii. In the past, prototheosis in dairy cattle was considered rare and the infection was associated with poor environmental conditions and inadequate milking hygiene. However, cases of prototecal mastitis are increasingly recognized as endemic throughout the world. Bovine mastitis is an inflammatory response of the udder, which is characterized by pathological changes in the mammary gland, an abrupt decrease in milk production, an increase in the count of somatic cells, and physiochemical changes in milk. Unlike human protothecosis, bovine mastitis can spread among farm animals. This contagion is favored by the fact that prototecal mastitis does not normally present evident clinical signs. Due to the high resistance of P. zopfii to conventional antimicrobial therapy, the best method for control is through early diagnosis and elimination of infected cows. As a consequence, protothecal bovine mastitis produces considerable economic losses throughout the world.
Pollution control with Prototheca
Due to the resistance of these algae to chlorination, antifungal and antibacterial treatments, it is important to adopt prevention and control measures in the main places of infection. Rice workers, livestock farms, fishermen, farmers, raw shellfish handlers, and aquarium personnel are especially at risk of exposure to Prototheca species. Mainly, the control of Prototheca becomes important in the livestock farms, where it supposes great economic losses. In addition, because prototheosis is a zoonosis, there is a possibility that it could become a public health problem. To assess the degree of environmental contamination, the presence of Prototheca can be analyzed in different types of sample (animal drinking water, milking equipment washing water, nipple swabs, food, bedding material, feces, etc.). Also, the control of foods of animal origin, such as milk and its derivatives, is of vital importance.
The diagnosis of protothecosis has traditionally been made by examining the morphological and biochemical characteristics of the isolated organism. However, these methods tend to generate equivocal results, and it has often been difficult to determine the Prototheca species causing the lesions. Recently, it has been shown that molecular techniques, such as polymerase chain reaction (PCR) using specific primers of species and genotypes, nucleotide sequencing of ribosomal DNA, proteomic analysis, and real-time PCR, are tools useful in the identification of Prototheca spp.
Tests offered by IVAMI:
- Detection of Prototheca spp. for isolation in culture, presumptive identification and quantification.
- Detection of Prototheca spp. by isolation in culture and molecular identification at the species level.
- Direct molecular detection of Prototheca spp.
- Molecular detection of Prototheca spp. and identification at the species level.
- Liquid sample suspicious or controlled (water, milk). A minimum of 250 ml or grams introduced in a sterile polypropylene (non-rigid plastic) container is recommended.
- Solid sample suspected or controlled (food, soil, feces ...). A minimum of about 100 g in a sterile polypropylene (non-rigid plastic) container is recommended.
- Swabs for surface sampling.
Conservation and shipment of the sample:
- Refrigerated (preferred) for less than 2 days.
Delivery of results:
- Detection by isolation in culture and presumptive identification: 3 days
- Detection by isolation in culture and molecular identification: 5 days.
- Molecular detection of Prototheca spp. Direct: 2 days
- Molecular detection and identification at the species level: 3 days
Cost of the test:
- Cost of the culture test: consult firstname.lastname@example.org.
- Cost of culture test and molecular identification: consult email@example.com.
- Cost of the molecular detection test: consult firstname.lastname@example.org.
- Cost of the detection and molecular identification test: consult email@example.com.