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

IntroductionPigeon 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 StatementPigeon 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.AimTo investigate the environmental prevalence of C. neoformans in South-eastern Nigeria and compare the isolates with other lineages by using molecular and microbiological tools.MethodologyA 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.ConclusionThis 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.

Nanoparticles as therapeutic options for treating multidrug-resistant bacteria: research progress, challenges, and prospects.

Resistance to antimicrobial agents has been alarming in recent years and poses a huge public health threat globally according to the WHO. The increase in morbidity and mortality resulting from microbial infections has been attributed to the emergence of multidrug-resistant microbes. Associated with the increase in multidrug resistance is the lack of new and effective antimicrobials. This has led to global initiatives to identify novel and more effective antimicrobial agents in addition to discovering novel and effective drug delivery and targeting methods. The use of nanoparticles as novel biomaterials to fully achieve this feat is currently gaining global attention. Nanoparticles could become an indispensable viable therapeutic option for treating drug-resistant infections. Of all the nanoparticles, the metals and metal oxide nanoparticles appear to offer the most promise and have attracted tremendous interest from many researchers. Moreover, the use of nanomaterials in photothermal therapy has received considerable attention over the years. This review provides current insight on antimicrobial resistance as well as the mechanisms of nanoparticle antibacterial activity. It offers an in-depth review of all the recent findings in the use of nanomaterials as agents against multi-resistant pathogenic bacteria. Also, nanomaterials that can respond to light stimuli (photothermal therapy) to kill microbes and facilitate enhanced drug delivery and release are discussed. Moreover, the synergistic interactions of nanoparticles with antibiotics and other nanomaterials, microbial adaptation strategies to nanoparticles, current challenges, and future prospects were extensively discussed.

The persistence of amoebosis caused by Entamoeba histolytica in Nigeria and the role of malnutrition.

Amoebosis, caused by the protozoan parasite Entamoeba histolytica is a gastrointestinal infection and the second leading cause of death from parasitic disease worldwide. The disease is endemic in many developing countries and kills over one hundred thousand persons annually. Adequate nutrition composed of macro- and micronutrients in their balanced proportions is central to effective gut immune response and the homeostasis of commensal organisms in the gastrointestinal tract. Entamoeba histolytica is a gut pathobiont that can exploit a shift in nutritional status to cause amoebosis, with extra-intestinal complications. Although undernutrition is rarely a public health concern in high income settings, bioavailability of functional nutrients remains suboptimal. On the other hand, nutrient deficiencies constitute a chronic challenge in very low-income regions. This study sought to review the pivotal influence of malnutrition on intact microbiota and functional immunity, as determinants of susceptibility to amoebosis in the Nigerian example of tropical regions. The dynamics of the infection such as possible coinfection with opportunistic pathogens were also, evaluated. Based on the available reports, we posit that amoebosis is a common tropical infection perpetuated by malnutrition following poor living standard including unhygienic environmental exposure.

Evaluation of physicochemical and antioxidant properties of two stingless bee honeys: a comparison with Apis mellifera honey from Nsukka, Nigeria.

OBJECTIVE: Several physical, biochemical and antioxidant properties of two Nigerian stingless bee honey varieties (Melipona sp. and Hypotrigona sp.) were compared with Apis mellifera honey using standard analytical procedures. RESULTS: The mean pH of Apis mellifera, Hypotrigona sp. and Melipona sp. honeys were 4.24 ± 0.28, 3.75 ± 0.11 and 4.21 ± 0.37 respectively. The mean moisture contents of the honeys were 11.74 ± 0.47, 17.50 ± 0.80, and 13.86 ± 1.06%. Honey samples from Hypotrigona sp. when compared with other honey samples had the highest mean total dissolved solids (370.01 ± 22.51 ppm), hydroxymethylfurfural (16.58 ± 0.37 mg/kg), total acidity (35.57 ± 0.42 meq/kg), protein content (16.58 ± 0.37 g/kg), phenol content (527.41 ± 3.60 mg/kg), and ascorbic acid (161.69 ± 6.70 mg/kg), antioxidant equivalent-ascorbic acid assay value (342.33 ± 0.78 mg/kg) as well as ferric reducing power (666.88 ± 1.73 µM Fe(II)/100 g) (p < 0.05). Several strong correlations were observed among some of the parameters of the honeys. This is the first study to compare the properties of Nigerian honey bees. Our results suggested that these honeys (specifically Hypotrigona sp. honey) is a good source of antioxidants comparable to A. mellifera honey.

Human papillomavirus oncogenic E6 protein regulates human ß-defensin 3 (hBD3) expression via the tumor suppressor protein p53.

Human ß-defensin-3 (hBD3) is an epithelial cell-derived innate immune regulatory molecule overexpressed in oral dysplastic lesions and fosters a tumor-promoting microenvironment. Expression of hBD3 is induced by the epidermal growth factor receptor signaling pathway. Here we describe a novel pathway through which the high-risk human papillomavirus type-16 (HPV-16) oncoprotein E6 induces hBD3 expression in mucosal keratinocytes. Ablation of E6 by siRNA induces the tumor suppressor p53 and diminishes hBD3 in HPV-16 positive CaSki cervical cancer cells and UM-SCC-104 head and neck cancer cells. Malignant cells in HPV-16-associated oropharyngeal cancer overexpress hBD3. HPV-16 E6 induces hBD3 mRNA expression, peptide production and gene promoter activity in mucosal keratinocytes. Reduction of cellular levels of p53 stimulates hBD3 expression, while activation of p53 by doxorubicin inhibits its expression in primary oral keratinocytes and CaSki cells, suggesting that p53 represses hBD3 expression. A p53 binding site in the hBD3 gene promoter has been identified by using electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP). In addition, the p63 protein isoform ΔNp63α, but not TAp63, stimulated transactivation of the hBD3 gene and was co-expressed with hBD3 in head and neck cancer specimens. Therefore, high-risk HPV E6 oncoproteins may stimulate hBD3 expression in tumor cells to facilitate tumorigenesis of HPV-associated head and neck cancer.

ISOLATION OF Cryptococcus neoformans FROM ENVIRONMENTAL SAMPLES COLLECTED IN SOUTHEASTERN NIGERIA.

Cryptococcosis caused by Cryptococcus neoformans is the second most common fungal opportunistic pathogen and a life-threatening infection with serious clinical manifestations especially in HIV/AIDS and other immunocompromised patients. In Nigeria, HIV/AIDS infection has reached an alarming level. Despite this, information on the presence of this fungus in clinical and environmental samples is very scanty in Nigeria and many other parts of Africa. We set out to evaluate the presence of Cryptococcus neoformans or C. gattiiin pigeon droppings obtained from Southeastern Nigeria. One hundred and seventy-seven samples of pigeon droppings from six sample types were collected. The area covered comprised of ten cities and other locations spanning across five States in Nigeria. Using established techniques, Cryptococcus neoformans was isolated from 39 of the 177 (22.0%) samples overall. No C. gattiiwas isolated. Most of the isolates (32.4%) were recovered from dovecotes (11 of 34) followed closely by samples taken from markets (31.8%; seven of 22) and least from the church (4.0%; one of 25). The highest isolation rate (38.9%) was found in samples from Enugu-Ezike(seven of 23) while the least came from Afikpo and the other locations each with 9.1% isolation rate. This is the first large-scale screening of Cryptococcus neoformans from pigeon droppings in Nigeria. The ecological and epidemiological significance of these findings are discussed.

Isolation of cryptococcus neoformans from environmental samples collected in Southeastern Nigeria / Isolamento de Cryptococcus neoformans de amostras ambientais coletadas no sudeste da Nigéria

SUMMARYCryptococcosis caused by Cryptococcus neoformansis the second most common fungal opportunistic pathogen and a lifethreatening infection with serious clinical manifestations especially in HIV/AIDS and other immunocompromised patients. In Nigeria, HIV/AIDS infection has reached an alarming level. Despite this, information on the presence of this fungus in clinical and environmental samples is very scanty in Nigeria and many other parts of Africa. We set out to evaluate the presence of Cryptococcus neoformansor C. gattiiin pigeon droppings obtained from Southeastern Nigeria. One hundred and seventy-seven samples of pigeon droppings from six sample types were collected. The area covered comprised of ten cities and other locations spanning across five States in Nigeria. Using established techniques, Cryptococcus neoformanswas isolated from 39 of the 177 (22.0%) samples overall. No C. gattiiwas isolated. Most of the isolates (32.4%) were recovered from dovecotes (11 of 34) followed closely by samples taken from markets (31.8%; seven of 22) and least from the church (4.0%; one of 25). The highest isolation rate (38.9%) was found in samples from Enugu-Ezike(seven of 23) while the least came from Afikpoand the other locations each with 9.1% isolation rate. This is the first large-scale screening of Cryptococcus neoformansfrom pigeon droppings in Nigeria. The ecological and epidemiological significance of these findings are discussed.

RESUMOA criptococose, causada por Cryptococcus neoformans, é o segundo patógeno fúngico oportunista mais comum em infecções com risco de vida e manifestações clínicas graves, especialmente em HIV/AIDS e outros pacientes imunocomprometidos. Na Nigéria, a infecção pelo HIV/AIDS atingiu um nível alarmante. Apesar disso, informações sobre a presença desses fungos em amostras clínicas e ambientais é muito escassa na Nigéria e em muitas outras partes da África. Propusemo-nos a avaliar a presença de Cryptococcus neoformansou C. gattiiem fezes de pombos obtidos do sudeste da Nigéria. Foram coletadas 177 amostras de fezes de pombos de seis localidades. A área coberta foi composta por dez cidades e outras localidades abrangendo cinco Estados na Nigéria. Usando técnicas estabelecidas, Cryptococcus neoformansfoi isolado de 39 do total de 177 (22,0%) amostras. Nenhuma amostra de C. gattiifoi isolada. A maioria dos isolados (32,4%) foi recuperada de pombais (11 de 34), seguido de perto por amostras colhidas em mercados (31,8%; 7 de 22) e por último na igreja (4,0%; 1 de 25). A maior taxa de isolamento (38,9%) foi encontrada em amostras de Enugu-Ezike (7 de 23), enquanto a menor taxa foi de Afikpo e os outros locais, cada um deles com taxa de isolamento de 9,1%. Esta é a primeira triagem em larga escala de Cryptococcus neoformansem fezes de pombos na Nigéria. A importância ecológica e epidemiológica destes achados é discutida.

Frequency, urinalysis and susceptibility profile of pathogens causing urinary tract infections in Enugu State, southeast Nigeria.

OBJECTIVE: This study was designed to determine the frequency and causative agent(s) of urinary tract infections (UTIs) in individuals with symptoms of urinary tract infections in Enugu State of Southeast Nigeria, and to determine the antibiotic susceptibility pattern of microbial agents isolated from urine culture. METHODS: The study involved 211 individuals (149 females and 62 males) clinically suspected for UTI. Urine samples were collected by the mid-stream 'clean catch' method and tested using standard procedures. Antibiotic susceptibility of the isolated pathogens was tested using the Kirby-Bauer technique according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. RESULTS: Microscopy of centrifuged urine samples showed 16 patients had pyuria while 54 had pus cells. Calcium oxalate crystals were found in 14 samples. Urinalysis performed with urine samples showed 17 had protein; seven were nitrite positive and three had moderate to high glucose concentration. Fifty-four urine samples (36.2%) from females and 12 (19.4%) from males showed significant growth upon culture. Gram stain and biochemical tests identified nine different organisms with Escherichia coli as the most common isolated species. Forty three randomly selected strains were further tested for their susceptibility against a panel of antibiotics. Thirty isolates (81.08%) were resistant to four or more antibiotics with the highest resistance shown by E. coli (76.67%). All the Gram- negative isolates were resistant to Ampicilox, Cefuroxime and Amoxicillin. CONCLUSION: Urinary tract infections were found more in females in the area under study. As found in other studies, E. coli was the most predominant isolate, although other organisms seem to be on the increase.

Frequency, urinllysis and susceptibility profil of pathogens causing urinary ttract infections in enugu state, southeast Nigeria / Perfil da frequencia, analise urinaria e suscetibilidade de patogenos causando infeccoes do trato urinario no estado de Enugu, Sudeste da Nigeria

Objective: This study was designed to determine the frequency and causative agent(s) of urinary tract infections (UTIs) in individuals with symptoms of urinary tract infections in Enugu State of Southeast Nigeria, and to determine the antibiotic susceptibility pattern of microbial agents isolated from urine culture. Methods: The study involved 211 individuals (149 females and 62 males) clinically suspected for UTI. Urine samples were collected by the mid-stream ‘clean catch' method and tested using standard procedures. Antibiotic susceptibility of the isolated pathogens was tested using the Kirby-Bauer technique according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: Microscopy of centrifuged urine samples showed 16 patients had pyuria while 54 had pus cells. Calcium oxalate crystals were found in 14 samples. Urinalysis performed with urine samples showed 17 had protein; seven were nitrite positive and three had moderate to high glucose concentration. Fifty-four urine samples (36.2%) from females and 12 (19.4%) from males showed significant growth upon culture. Gram stain and biochemical tests identified nine different organisms with Escherichia coli as the most common isolated species. Forty three randomly selected strains were further tested for their susceptibility against a panel of antibiotics. Thirty isolates (81.08%) were resistant to four or more antibiotics with the highest resistance shown by E. coli (76.67%). All the Gram- negative isolates were resistant to Ampicilox, Cefuroxime and Amoxicillin. Conclusion: Urinary tract infections were found more in females in the area under study. As found in other studies, E. coli was the most predominant isolate, although other organisms seem to be on the increase.

Objetivo: O estudo teve como objetivo determinar a frequência e agentes causadores das infecções do trato urinário (UTIs) em indivíduos com sintomas desta infecção no estado de Enugu, Sudeste da Nigéria e determinar a suscetibilidade antibiótica dos agentes microbianos isolados de cultura da urina. Métodos: O estudo envolveu 211 indivíduos (149 mulheres e 62 homens) clinicamente suspeitos para UTI. Amostras urinárias foram coletadas pelo método de meia corrente “clean catch” e testados por procedimentos standards. Suscetibilidade aos antibióticos dos patógenos isolados foi testada usando a técnica de Kirby-Bauer e de acordo com as diretrizes do “Clinical and Laboratory Standards Institute” (CLSI). Resultados: Microscopia das amostras de urina centrifugadas mostraram que 16 pacientes tinham piúria enquanto que 54 tinham células do pus. Cristais de oxalato de cálcio foram encontrados em 14 amostras. Análise de amostras da urina mostraram que 17 tinham proteína; sete eram positivas para nitrito e três tinham concentração de glicose de moderada para alta. Cinquenta e quatro amostras de urina (36.2% de mulheres) e 12 (19.45% de homens) mostraram crescimento significante após cultura. Coloração pelo Gram e testes bioquímicos identificaram nove organismos diferentes com a Escherichia coli sendo a mais comum das espécies isoladas. Quarenta e três espécies selecionadas ao acaso foram testadas posteriormente para sua suscetibilidade contra um painel de antibióticos. Trinta amostras isoladas (81.08%) foram resistentes a quatro ou mais antibióticos sendo que a maior resistência foi da E. coli (76.67%). Todas as amostras Gram negativas isoladas foram resistentes a Ampicilox, Cefuroxime e Amoxicilina.

Development of a 96-well catheter-based microdilution method to test antifungal susceptibility of Candida biofilms.

BACKGROUND: Candida biofilms, which are often associated with device-related infections, including catheter-related bloodstream infections, are resistant to commonly used antifungal agents. Current microtitre (96-well) plate-based methods to determine the antifungal susceptibility of these biofilms do not involve clinically relevant substrates (e.g. catheters), and are not well suited for evaluating the surface topography and three-dimensional architecture of biofilms. We describe a simple, reproducible catheter-based microtitre plate method to form biofilms and evaluate their antifungal susceptibility. METHODS: Biofilms were formed by Candida species on 5 mm catheter discs placed in microtitre plates and quantified using metabolic conversion of a formazan dye (XTT). The morphology, surface topography and three-dimensional architecture of these biofilms were evaluated by fluorescence, confocal scanning laser and scanning electron microscopy, respectively. The optimized XTT method was used to evaluate the antifungal susceptibility of formed Candida biofilms to fluconazole, voriconazole, itraconazole and anidulafungin. RESULTS: Maximum XTT activity was achieved within 90 min. All tested Candida strains formed robust biofilms on catheter discs at both 24 and 48 h (P = 0.66). Biofilms exhibited typical gross morphology, surface topography and architecture, and no difference in biofilm thickness (P = 0.37). The three tested azoles were not active against the biofilms (MIC ≥ 64 mg/L), but anidulafungin possessed potent activity against them (MIC = 0.063-0.125 mg/L). CONCLUSIONS: The developed method is simple, rapid and reproducible, and requires relatively small amounts of drug. It can be used to perform both high-resolution microscopic analysis of the topography and architecture of biofilms, and evaluation of their antifungal susceptibility.

Dermatophytoses in domesticated animals.

Dermatophytes are among the most frequent causes of ringworm infections in domesticated animals. They are known to serve as reservoirs of the zoophilic dermatophytes and these infections have important zoonotic implication. In Nigeria and probably West Africa, there are not many studies on the incidence of dermatophytosis in domesticated animals. In the current study, 538 domesticated animals with clinically suggestive lesions were investigated for dermatophytes. Identification of dermatophyte species was performed by macro- and micro morphological examination of colonies and by biochemical methods. In the cases of isolates that had atypical morphology and/or biochemical test results, the rDNA internal transcribed spacer region 2 (ITS 2) sequencing was performed. Out of this number, 214 (39.8%) were found to be colonized by a variety of ten species of dermatophytes. M. canis was the most frequently isolated species (37.4%), followed by T. mentagrophytes (22.9%) and T. verrucosum (15.9%). M. persicolor and T. gallinae were jointly the least species isolated with a frequency of 0.55% respectively. The recovery of dermatophyte isolates previously shown to be common etiological agents of dermatophytosis especially from children in the same region suggests that animal to human transmission may be common. Possible implications and recommendations are discussed.

Dermatophytoses in domesticated animals / Dermatofitoses em animais domésticos

Dermatophytes are among the most frequent causes of ringworm infections in domesticated animals. They are known to serve as reservoirs of the zoophilic dermatophytes and these infections have important zoonotic implication. In Nigeria and probably West Africa, there are not many studies on the incidence of dermatophytosis in domesticated animals. In the current study, 538 domesticated animals with clinically suggestive lesions were investigated for dermatophytes. Identification of dermatophyte species was performed by macro- and micro morphological examination of colonies and by biochemical methods. In the cases of isolates that had atypical morphology and/or biochemical test results, the rDNA internal transcribed spacer region 2 (ITS 2) sequencing was performed. Out of this number, 214 (39.8 percent) were found to be colonized by a variety of ten species of dermatophytes. M. canis was the most frequently isolated species (37.4 percent), followed by T. mentagrophytes (22.9 percent) and T. verrucosum (15.9 percent). M. persicolor and T. gallinae were jointly the least species isolated with a frequency of 0.55 percent respectively. The recovery of dermatophyte isolates previously shown to be common etiological agents of dermatophytosis especially from children in the same region suggests that animal to human transmission may be common. Possible implications and recommendations are discussed.

Dermatófitos estão entre as causas mais frequentes de infecções tipo larva migrans em animais domésticos. Eles são conhecidos como reservatórios de dermatófitos zoófilos e estas infecções têm implicações zoonóticas importantes. Na Nigéria e provavelmente no Oeste da África não existem muitos estudos sobre a incidência de dermatofitose em animais domésticos. No presente estudo, 538 animais domésticos com lesões clinicamente sugestivas foram investigados para dermatófitos. Identificação de espécies de dermatófito foi feita pelo exame macro e microscópico morfológico de colônias e por métodos bioquímicos. No caso de isolados com morfologia atípica e/ou resultados de testes bioquímicos, sequenciamento da região 2 transcrita (ITS-2) do rDNA foi feita. Dos casos, 214 (39,8 por cento) foram colonizados por uma variedade de 10 espécies de dermatófitos. M. canis foi a espécie isolada mais frequente (37,4 por cento) seguida por T. mentagrophytes (22,9 por cento) e de T. verrucosum (15,9 por cento). M. persicolor e T. gallinae foram juntos as menos frequentes espécies isoladas (0,55 por cento respectivamente). O isolamento destes dermatófitos que são agentes etiológicos comuns de dermatofitoses especialmente de crianças da mesma região sugere que a transmissão de animais para humanos possa ser comum. Possíveis implicações e recomendações são discutidas.

Justification for the use of Ocimum gratissimum L in herbal medicine and its interaction with disc antibiotics.

BACKGROUND: The ethanolic extract of the leaves of Ocimium gratisimum L. (Lamiaceae), used in traditional medicine for the treatment of several ailments such as urinary tract, wound, skin and gastrointestinal infections, was evaluated for its antibacterial properties against four clinical bacteria isolates namely: Escherichia coli, Proteus mirabilis, Staphylococcus aureus and Pseudomonas aeruginosa and the antifungal properties using a clinical isolate of Candida albicans. A typed bacterium of Escherichia coli ATCC 11775 and another typed fungal strain of Candida albicans (ATCC 90028) were also included. The study also intended to verify if the concomitant administration of conventional antibiotics with Ocimium gratisimum which is normally taken as food (spice) will negatively affect its activity. METHODS: The agar diffusion method was used to test the in vitro activity of the plant extract. The interaction of the plant extract with some disc antibiotics namely: ciprofloxacin, septrin, streptomycin, ampicillin, nystatin and ketoconazole was tested using the agar overlay inoculum susceptibility disc method. Phytochemical analysis of the extract was performed following established methods. RESULTS: The extract showed good but varying in vitro activities against all the isolates tested. While ampicillin showed synergistic interaction with the plant extract against clinical isolates of E. coli and P. mirabilis, septrin was synergistic against the clinical isolate of E. coli only. Similarly, the activity of the extract against C. albicans isolate was synergistic with ketoconazole and nystatin. CONCLUSION: The study has validated the folkloric use of O. gratissimum in traditional medicinal practice and goes further to show that the use of this plant material as food spice may not really threaten the efficacy of some conventional antibiotics that may have been taken concomitantly with it as is the popular belief in the practice of herbal medicine in local/rural communities of many countries in the world.