Multi-locus sequence typing reveals genotypic similarity in Nigerian Cryptococcus neoformans AFLP1/VNI of environmental and clinical origin

Introduction Pigeon droppings are among the major environmental sources of Cryptococcus neoformans AFLP1/VNI, from where the organism infects susceptible humans and animals resulting in cryptococcosis. Until now, C. neoformans AFLP1B/VNII was the only molecular type reported in Nigeria. Effective clinical treatment of this infection has occasionally been stymied by the emergence of antifungal non-susceptible, and resistant strains of C. neoformans AFLP1/VNI. Hypothesis/Gap Statement Pigeon droppings harbour C. neoformans and HIV/AIDS patients are among the susceptible population to develop cryptococcal infection. Epidemiological data on cryptococcal prevalence is limited in Nigeria. Aim To investigate the environmental prevalence of C. neoformans in South-eastern Nigeria and compare the isolates with other lineages by using molecular and microbiological tools. Methodology A total of 500 pigeon droppings and 300 blood samples of HIV/AIDS patients were collected, respectively, from five market squares and three tertiary healthcare centres within the Nsukka area of South-eastern Nigeria. The antifungal susceptibility of the C. neoformans isolates to amphotericin B, fluconazole, 5-fluorocytosine, itraconazole, voriconazole, posaconazole, and isavuconazole was investigated based on the CLSI M27-A3 protocol. Yeasts were identified by MALDI-TOF MS, thereafter Cryptococcus MLST was performed according to the International Society for Human and Animal Mycology (ISHAM) consensus scheme. Results C. neoformans was recovered from 6 (1.2 %) pigeon droppings and 6 (2 %) blood cultures of HIV/AIDS patients. Molecular analyses indicated that all cryptococcal isolates belong to serotype A and the AFLP1/VNI molecular type with sequence type (ST)32. Infection with C. neoformans was independent of sex and age of the patients investigated. All C. neoformans isolates were susceptible to the seven antifungal agents. Conclusion This is the first report on the prevalence of C. neoformans AFLP1/VNI (ST32) in environmental and clinical samples from Nigeria. The antifungal susceptibility indicates that antifungal resistance by C. neoformans is yet a rare occurrence in Nigeria.

Establishment of Rat Models of Different Pathological Types of Hypersensitivity Pneumonitis Using Pigeon Droppings

<b><i>Background:</i></b> The pathogenesis and pulmonary histopathological characteristics of hypersensitivity pneumonitis (HP) are not yet fully understood. Therefore, we established animal models of HP of different stages, aiming to provide support for research on this disease. <b><i>Methods:</i></b> We established rat models of pigeon breeder’s lung of different pathological types by creating freeze-dried allergen powder from fresh pigeon feathers, dander, and other droppings. Freeze-dried allergen powder suspensions of pigeon droppings were used to establish 2 rat models of HP, one by aerosol inhalation and one by airway instillation, and the rats were sacrificed after different lengths of time to observe the pathological changes in their lung tissues. <b><i>Results:</i></b> By the 40th week after allergen inhalation, granulomas were the main changes in the model, without fibrotic changes. When using airway instillation to establish the model, at the 20th week, group 1 (low dose + twice/week) and group 2 (medium dose + twice/week) showed granuloma changes, but no fibrosis; group 3 (high dose + once/week) and group 4 (high dose + twice/week) both showed obvious pulmonary fibrotic changes, but the death rate of rats in group 4 was greater. <b><i>Conclusions:</i></b> Both aerosol inhalation and airway instillation of freeze-dried pigeon allergen powder can successfully establish an HP model. The airway instillation method can cause pulmonary fibrotic changes in a short time, and the pulmonary pathological changes of animal models manifest with an obvious time-dose effect.

Gastrointestinal helminths and coccidia infestation of city pigeons (Columba livia forma urbana) on selected monuments in Szczecin

City pigeons (Columba livia forma urbana) are an integral part of the urbanized environment. However, they host parasites that can be passed zoonotically to humans and to domestic and wild birds. The aim of the study was to establish the current prevalence of nematode and coccidians infestation among city pigeons roosting in old buildings in Szczecin. The pigeon kits, comprising city pigeons and stray breeding pigeons which had not returned to their lofts, numbered from 45 to 60 individuals. Samples of pigeon droppings were tested qualitatively by the flotation method and then quantitatively by the McMaster technique. Oocyst culture was also performed in a humid chamber at a temperature of 24–26° C. The oocytes were identified using standard keys. Two roundworm families were identified, Ascaridia spp. (two species) and Capillaria spp., and three coccidian species: Eimeria labbeana, E.columbarum and E. columbae. Overall, the extent of infection was approximately 100% throughout the study area, but varied depending on the place of origin of the material. The prevalence of worms with eggs ranged from 9% to 16% for Ascaris spp. and 24–41% for Capillaria spp. The maximum OPG values, i.e. the number of oocytes per gram of faeces, were 360 for Ascaris spp. and 1134 for Capillaria spp; these indicate the intensity of infection. The most frequently observed species was Eimeria labbeana, whose prevalence was found to range from 82% to 96% depending on location, with a mean infection intensity of 1130–1870 OPG. The prevalence of the other two Eimeria species was also high, with respectively values of 56–77% for E. columbarum and 32–43% for E. columbae, depending on the location. The mean OPG values for the species were 430–1770 and 29–52, respectively. Maximum OPG values were 223,000 for E. labbeana, 54,000 for E. columbarum and 830 for E. columbae.

Genomic epidemiology of a Cryptococcus neoformans case cluster in Glasgow, Scotland, 2018

In 2018, a cluster of two cases of cryptococcosis occurred at the Queen Elizabeth University Hospital (QEUH) in Glasgow, Scotland (UK). It was postulated that these cases may have been linked to pigeon droppings found on the hospital site, given there have been previous reports of Cryptococcus neoformans associated with pigeon guano. Although some samples of pigeon guano taken from the site yielded culturable yeast from genera related to Cryptococcus, they have since been classified as Naganishia or Papiliotrema spp., and no isolates of C. neoformans were recovered from either the guano or subsequent widespread air sampling. In an attempt to further elucidate any possible shared source of the clinical isolates, we used whole-genome sequencing and phylogenetic analysis to examine the relationship of the two Cryptococcus isolates from the QEUH cases, along with two isolates from sporadic cases treated at a different Glasgow hospital earlier in 2018. Our work demonstrated that these four clinical isolates were not clonally related; while all isolates were from the VNI global lineage and of the same mating type (MATα), the genotypes of the two QEUH isolates were separated by 1885 base changes and belonged to different sub-lineages, recently described as the intercontinental sub-clades VNIa-93 and VNIa-5. In contrast, one of the two sporadic 2018 clinical isolates was determined to belong to the VNIb sub-lineage and the other classified as a VNIV/VNI hybrid. Our work demonstrated that the two 2018 QEUH isolates and the two prior C. neoformans clinical isolates were all genetically distinct. It was not possible to determine whether the QEUH genotypes stemmed from independent sources or from the same source, i.e. pigeons carrying different genotypes, but it should be noted that whilst members of allied genera within the Tremellomycetes were isolated from the hospital environment, there were no environmental isolations of C. neoformans.

Predominance of Cryptococcus neoformans Var. grubii in Ahvaz, Molecular Identification and Evaluation of Virulence Factors

Background: Cryptococcus neoformans is an encapsulated yeast pathogen with worldwide distribution, and the highest incidence of cryptococcosis was attributed to C. neoformans (var. grubii. The pathogenicity of Cryptococcus species is associated with several factors, including capsule and melanin production, growth at 37 ºC, and secretion of extracellular enzymes. Objectives: The present study aimed to isolate and identify Cryptococcus species from pigeon guano in Ahvaz, Iran and investigate important virulence factors in the isolates. Methods: Seventy-three isolates of C. neoformans var. grubii were identified based on classical and molecular microbiology methods. Capsule size was measured by the grow yeasts in the presence of 5% CO2. Specific media demonstrated the activity of extracellular enzymes (phospholipase, hemolysin, proteinase, esterase, urease, catalase, and gelatinase). Besides, melanin production was evaluated by the niger seed agar medium. Results: Two hundred and seventeen samples were examined for the presence of Cryptococcus over 165 days in Ahvaz. All tested isolates were contained capsules with variable sizes under 5% CO2 concentration. Moreover, 100% of isolates were produced extracellular enzymes (urease, hemolysin, and catalase), whereas no proteinase and gelatinase activities were observed among isolates. Furthermore, most isolates had phospholipase (93.1%) and esterase activities (86.3%). Also, melanin was produced by all of the isolates. Conclusions: Although two methods were used for recovery of Cryptococcus, only Cryptococcus was isolated from pigeon guano, and swabs from the cage walls were negative. Cryptococcus neoformans var. grubii was the only species from pigeon droppings from Ahvaz with more pathogenic factors. Owing to the high pathogenicity of the isolates, the frequency of the disease is expected to be higher.

Primary Cutaneous Cryptococcosis: A Rare Masquerading Presentation of Cryptococcus Infection

The yeast, Cryptococcus is widely present in the environment. Its main portal of entry is the respiratory tract. Clinical and experimental evidences indicate that cryptococcosis is usually a reactivation of a dormant infection. They have been recovered from soil contaminated with avian excreta, especially pigeon droppings and from decaying wood, fruits, vegetables and dust. Cryptococcusmay be found on skin of healthy subjects without causing any manifestation but it is known to cause life threatening infection in immunocompromised hosts. Primary Cutaneous Cryptococcosis (PCC) is an uncommon condition which is characterised by localised cryptococcal skin eruptions without dissemination to internal organs. Clinicians are aware of the typical presentation of cryptococcal infections occurring mostly in immunocompromised patients. Rare manifestations like PCC may go unnoticed leading to prolonged morbidity and health care cost. The present article is a case report of PCC in a 39-year-old immunocompetent male who presented with two months history of scattered erythematous indurated papules and plaques on his right foot, arm and abdominal region developed after having suffered minor injury at the cement factory. The patient was started on fluconazole to which he responded well.

Occurrence and Distribution of Cryptococcus Species in Environmental Sources from Lower Assam Belt of India

Cryptococcus is a non-motile, gram positive, non-fermenting Basidiomycetous encapsulated yeast like fungus that causes respiratory, neurological and other systemic diseases in both humans and animals. Present study delineates the possible distribution of Cryptococcus species in pigeon droppings, excreta of other avian species, eucalyptus tree and contaminated soil specimens collected from different geographical co-ordinates of six geographical regions of the lower Brahmaputra Valley of Assam, India. The fungi were isolated through conventional methods of Sabouraud Dextrose Agar (SDA) and Bird Seed Agar (BSA) media and identified through negative staining of capsule as well as performing classical bio-chemical tests. Identity of the isolates was further confirmed through sequence analysis of ITS-1 and ITS-4 region of the 18S rDNA. Two pathogenic species of Cryptococcus were isolated from 67 (15.40%) of the 435 specimens. Of these positive isolates 41 (9.43%) belonged to Naganishia albida (Cryptococcus albidus) while 26 (5.98%) represented Papiliotrema laurentii (Cryptococcus laurentii). Both the species were recovered from 58 (18.35%) dry and 9 (7.56%) moist specimens. The percentage of prevalence of Naganishia albida in dry and moist specimens were 35 (11.07%) and 6 (5.04%) respectively. Contrary to this, the percentage of prevalence of Papiliotrema laurentii in dry and moist were 23 (7.28%) and 3 (2.52%) respectively. The findings indicate that Cryptococcus species have established a better ecological sustenance in dry specimens than moist. The findings of the investigation demonstrated that the prevalence of Cryptococcus albidus in attics, dovecotes / houses of pigeon fanciers, contaminated soil, eucalyptus tree and droppings of other birds were 11(12.36%) out of 89, 23(14.11%) of 163,2(3.23%) of 62,4(7.84%) of 51 and only 1(1.43%) out of 7 specimens respectively. The recovery of Papiliotrema laurentii in the above specimens were 3(3.37%), 20(12.27%), 1(1.61%), 1(1.96%) and 1(1.42%) respectively. The findings revealed that the prevalence of Naganishia albida is more than that of Papiliotrema laurentii in natural substrates. The notorious pathogenic fungi, Cryptococcus neoformans could not be isolated, indicative of the fact that the region selected for the study is not environmentally favorable for growth and sustenance of the species. Findings of the study clearly demonstrate the ecological and epidemiological significance of the non-neoformans species of the genus cryptococcus that needs further comprehensive studies to access the prevalence of the genus from public health point of view.

Weathering Processes and Mechanisms Caused by Capillary Waters and Pigeon Droppings on Porous Limestones

This investigation studies the physical and chemical effect of salt weathering on biocalcarenites and biocalcrudites in the Basilica of Our Lady of Succour (Aspe, Spain). Weathering patterns are the result of salty rising capillary water and water lixiviated from pigeon droppings. Surface modifications and features induced by material loss are observable in the monument. Formation of gypsum, hexahydrite, halite, aphthitalite and arcanite is associated with rising capillary water, and niter, hydroxyapatite, brushite, struvite, weddellite, oxammite and halite with pigeon droppings. Humberstonite is related to the interaction of both types of waters. Analysis of crystal shapes reveals different saturation degree conditions. Single salts show non-equilibrium shapes, implying higher crystallisation pressures. Single salts have undergone dissolution and/or dehydration processes enhancing the deterioration process, particularly in the presence of magnesium sulphate. Double salts (humberstonite) have crystals corresponding to near-equilibrium form, implying lower crystallisation pressures. This geochemical study suggests salts precipitate via incongruent reactions rather than congruent precipitation, where hexahydrite is the precursor and limiting reactant of humberstonite. Chemical dissolution of limestone is driven mainly by the presence of acidic water lixiviated from pigeon droppings and is a critical weathering process affecting the most valuable architectural elements present in the façades.

Screening and Selection of Cryptococcus Neoformans Using Urease Activity and Temperature Range

Pigeon dropping has been known to be a reservoir for Cryptococcus neoformans. In this research, attempt has been made to screen and select for this life threatening organism using temperature (thermotolerance) and urease test sequentially. Fourteen (14) samples from pigeon droppings were collected from three markets in Benin City. Isolates obtained from these samples were subjected to urease and temperature test. The results showed that 10 isolates that were positive for temperature were equally positive for the urease tests, with highest growth rate 37°C recorded for samples obtained from New Benin (NB3). This experiment demonstrates the possibility of isolating C. neoformans at minimum temperature of 37°C thermotolerance and urease activity tests. Pathogenicity studies are recommended to further understand host – pathogen relationships. Keywords: Cryptococcus neoformans, Pathogenic, Urease, Thermotolerance, Isolates