Antifungal susceptibilities, biofilms, phospholipase and proteinase activities in the Candida rugosa complex and Candida pararugosa isolated from tertiary teaching hospitals.

PURPOSE: Non-albicansCandida species have emerged as fungal pathogens that cause invasive infections, with many of these species displaying resistance to commonly used antifungal agents. This study was confined to studying the characteristics of clinical isolates of the C. rugosa complex and C. pararugosa species. METHODOLOGY: Seven isolates of the C. rugosa complex and one isolate of C. pararugosa were obtained from two tertiary referral hospitals in Malaysia. Their antifungal susceptibilities, biofilm, proteinase, phospholipase, esterase and haemolysin activities were characterized. Biofilms were quantified using crystal violet (CV) and tetrazolium (XTT) reduction assays at 1.5, 6, 18, 24, 48 and 72 h.Results/Key findings. The E-test antifungal tests showed that both species have elevated MICs compared to C. albicans and C. tropicalis. The highest biomass was observed in one of the C. rugosa isolates (0.237), followed by C. pararugosa (0.206) at 18 h of incubation. However, the highest bioactivity was observed in the C. rugosa ATCC 10571 strain at 24 h (0.075), followed by C. pararugosa at 48 h (0.048) and the same C. rugosa strain at 24 h (0.046), with P<0.05. All isolates exhibited high proteinase activity (+++) whereas six isolates showed very strong esterase activity (++++). All the isolates were alpha haemolytic producers. None of the isolates exhibited phospholipase activity. CONCLUSION: Elevated MICs were shown for the C. rugosa complex and C. pararugosa for commonly used antifungal drugs. Further studies to identify virulence genes involved in the pathogenesis and genes that confer reduced drug susceptibility in these species are proposed.

Selection of reference genes for quantitative studies in acute myeloid leukaemia.

INTRODUCTION: Quantitative polymerase chain reaction (qPCR) is commonly used in the investigation of acute myeloid leukaemias (AML). Stable reference genes (RG) are essential for accurate and reliable reporting but no standard method for selection has been endorsed. MATERIALS AND METHODS: We evaluated simple statistics and published model-based approaches. Multiplex-qPCR was conducted to determine the expression of 24 candidate RG in AMLs (N=9). Singleplex-qPCR was carried out on selected RG (SRP14, B2M and ATP5B) and genes of interest in AML (N=15) and healthy controls, HC (N=12). RESULTS: RG expression levels in AML samples were highly variable and coefficient of variance (CV) ranged from 0.37% to 10.17%. Analysis using GeNorm and Normfinder listed different orders of most stable genes but the top seven (ACTB, UBE2D2, B2M, NF45, RPL37A, GK, QARS) were the same. In singleplex-qPCR, SRP14 maintained the lowest CV in AML samples. B2M, one of most stable reference genes in AML, was expressed near significantly different in AML and HC. GeNorm selected ATP5B+SRP14 while Normfinder chose SRP14+B2M as the best two RG in combination. The median expressions of combined RG genes in AML compared to HC were less significantly different than individually implying smaller expression variation after combination. Genes of interest normalised with RG in combination or individually, displayed significantly different expression patterns. CONCLUSIONS: The selection of best reference gene in qPCR must consider all sample sets. Model-based approaches are important in large candidate gene analysis. This study showed combination of RG SRP14+B2M was the most suitable normalisation factor for qPCR analysis of AML and healthy individuals.

Antimicrobial susceptibilities and random amplified polymorphic DNA-PCR fingerprint characterization of Candida glabrata, Candida parapsilosis and Candida rugosa from two major hospitals in Kuala Lumpur, Malaysia.

The purpose of this study is to characterize 3 non-albicans Candida spp. that were collected from two major hospitals in a densely populated area of Kuala Lumpur for their susceptibilities to azole and genetic background. Fifteen non-albicans Candida clinical isolates in two major hospitals in Kuala Lumpur area of Malaysia were collected by convenience sampling during 2007 and 2010. The genetic diversity of 15 non-albicans Candida species comprising C. glabrata (n = 5), C. parapsilosis (n = 5) and C. rugosa (n = 5) were assessed by RAPD-PCR typing. Strains were initially identified using biochemical tests and CHROMagar Candida medium. Fluconazole and voriconazole susceptibilities were determined by E-test method. Commercial kits were used for DNA extraction and amplification with RAPD primers (OPA02, OPA03 and OPA08). PCR conditions were optimized and simultaneous identification was possible by agarose gel electrophoresis of PCR products and the bands obtained were analyzed using BioNumerics Applied Maths v.6.6 software. The RAPD primers used in this study generated 100% polymorphic profile. Cluster analysis using the RAPD-PCR profile showed 12.5-25% similarity among the strains. The genetic diversity was based on the strain susceptibility towards both the azoles, site of isolation and place according to their unique banding patterns. In contrast, strains susceptible to azoles were found to be genetically similar with clonal dissimilarity. The use of OPA02, OPA03 and OPA08 primers in differentiating non-albicans Candida spp. underscores the higher resolution of RAPD-PCR as a reliable tool for strain/species level differentiation.

Antimicrobial susceptibilities and random amplified polymorphic DNA-PCR fingerprint characterization of Candida glabrata, Candida parapsilosis and Candida rugosa from two major hospitals in Kuala Lumpur, Malaysia

@#The purpose of this study is to characterize 3 non-albicans Candida spp. that were collected from two major hospitals in a densely populated area of Kuala Lumpur for their susceptibilities to azole and genetic background. Fifteen non-albicans Candida clinical isolates in two major hospitals in Kuala Lumpur area of Malaysia were collected by convenience sampling during 2007 and 2010. The genetic diversity of 15 non-albicans Candida species comprising C. glabrata (n = 5), C. parapsilosis (n = 5) and C. rugosa (n = 5) were assessed by RAPD-PCR typing. Strains were initially identified using biochemical tests and CHROMagar Candida medium. Fluconazole and voriconazole susceptibilities were determined by E-test method. Commercial kits were used for DNA extraction and amplification with RAPD primers (OPA02, OPA03 and OPA08). PCR conditions were optimized and simultaneous identification was possible by agarose gel electrophoresis of PCR products and the bands obtained were analyzed using BioNumerics Applied Maths v.6.6 software. The RAPD primers used in this study generated 100% polymorphic profile. Cluster analysis using the RAPD-PCR profile showed 12.5-25% similarity among the strains. The genetic diversity was based on the strain susceptibility towards both the azoles, site of isolation and place according to their unique banding patterns. In contrast, strains susceptible to azoles were found to be genetically similar with clonal dissimilarity. The use of OPA02, OPA03 and OPA08 primers in differentiating non-albicans Candida spp. underscores the higher resolution of RAPD-PCR as a reliable tool for strain/species level differentiation.

Successful Diagnosis of Intestinal Mycobacterium avium Complex Infection in a Kidney Transplant Recipient Using Nasogastric Aspirate Culture: A Case Report.

Intestinal Mycobacterium avium complex (MAC) infections are rare and can be challenging to diagnose. We describe a case of intestinal MAC infection in a kidney transplant recipient with 5 months of unexplained weight loss and abdominal pain who developed intestinal obstruction. Esophagoduodenoscopy with biopsies was performed but was nondiagnostic. Intestinal MAC was diagnosed via nasogastric aspirate culture results. The patient's symptoms rapidly improved after initiation of appropriate treatment, but he later died of aspiration pneumonia and candidemia.

The role of host microfilaments and microtubules during opsonin-independent interactions of Cryptococcus neoformans with mammalian lung cells.

The purpose of this investigation was to characterise the interactions of Cryptococcus neoformans with mammalian host alveolar epithelial cells and alveolar macrophages, with emphasis on the roles of the cryptococcal capsule and the host cell cytoskeletons. The adherence and internalisation of C. neoformans into mammalian lung cells and the roles of host cell cytoskeletons in host-pathogen interactions were studied using in vitro models coupled with a differential fluorescence assay, fluorescence staining, immunofluorescence and drug inhibition of actin and microtubule polymerisation. Under conditions devoid of opsonin and macrophage activation, C. neoformans has a high affinity towards MH-S alveolar macrophages, yet associated poorly to A549 alveolar epithelial cells. Acapsular C. neoformans adhered to and internalised into the mammalian cells more effectively compared to encapsulated cryptococci. Acapsular C. neoformans induced prominent actin reorganisation at the host-pathogen interface in MH-S alveolar macrophages, but minimally affected actin reorganisation in A549 alveolar epithelial cells. Acapsular C. neoformans also induced localisation of microtubules to internalised cryptococci in MH-S cells. Drug inhibition of actin and microtubule polymerisation both reduced the association of acapsular C. neoformans to alveolar macrophages. The current study visualises and confirms the interactions of C. neoformans with mammalian alveolar cells during the establishment of infection in the lungs. The acapsular form of C. neoformans effectively adhered to and internalised into alveolar macrophages by inducing localised actin reorganisation, relying on the host's actin and microtubule activities.

Immunoproteomic analysis of antibody response to cell wall-associated proteins of Candida tropicalis.

AIMS: This study was conducted to identify antigenic proteins of Candida tropicalis that are targeted by the host immune system. METHODS AND RESULTS: An immunoproteomic approach was used to discover antigens from cell wall of C. tropicalis that were recognized by sera from experimentally infected mice. This resulted in the identification of twelve distinct proteins, of which ten have been previously reported as antigens of Candida albicans. For the remaining two proteins, Idh2p has been described as an antigen of Candida parapsilosis, whereas Kgd2p is revealed for the first time as an antigenic protein for Candida species. These two antigens were expressed as recombinant proteins in Escherichia coli and were shown to be specifically recognized by sera from infected host on Western blot. CONCLUSIONS: The present work investigated immunoproteome of C. tropicalis and identified several biomarker candidate antigens, with Kgd2p as a novel immunogenic protein that could be associated with pathogenesis of C. tropicalis. SIGNIFICANCE AND IMPACT OF THE STUDY: Findings from this study help to improve current understanding on host response to C. tropicalis infection and provide new insights into immune-pathogenesis of C. tropicalis. Besides, the immunogenic proteins could be considered as targets for the development of immunodiagnostic assay and/or vaccine.

Comparison of nested and ELISA based polymerase chain reaction assays for detecting Chlamydia trachomatis in pregnant women with preterm complications.

Identification of pregnant women infected with Chlamydia trachomatis is essential to allow early antibiotic treatment in order to prevent adverse pregnancy outcomes. In this study, two nucleic acid amplification tests (NAAT) namely nested PCR (BioSewoom, Korea) and Amplicor CT/NG (Roche Diagnostic, USA) were evaluated in terms of sensitivity and specificity for the detection of C. trachomatis DNA in pregnant women with preterm complications. A cross-sectional study was carried out in two public hospitals in Southern Selangor, Malaysia. Endocervical swabs obtained were subjected to DNA amplification using nested PCR (BioSewoom, Korea) and Amplicor CT/NG (Roche Diagnostic, USA). A total of 83 endocervical swabs obtained from pregnant women of less than 37 weeks gestation and presented with preterm complications were subjected to chlamydial DNA detection using both assays. The study shows that Amplicor CT/NG assay is more effective in the detection of C. trachomatis DNA from endocervical swabs compared to Biosewoom nested PCR kit. Agreement between the two assays were poor (kappa=0.094) with nested PCR showing a low sensitivity of 10.81% and a 97.83% specificity when compared to Amplicor CT/NG. The results obtained indicated that BioSewoom nested PCR was less sensitive than Amplicor CT/ NG for detecting C. trachomatis in endocervical specimens and that another more reliable test is required for confirmatory result.

Identification of immunogenic proteins of Candida parapsilosis by serological proteome analysis.

AIMS: Systemic candidiasis is the leading fungal bloodstream infection, and its incidence has been on the rise. Recently, Candida parapsilosis has emerged as an increasingly prevalent fungal pathogen, but little is known about its antigenic profile. Hence, the current work was performed to discover immunogenic proteins of C. parapsilosis using serological proteome analysis. METHODS AND RESULTS: Cell wall proteins extracted from C. parapsilosis were resolved by two-dimensional electrophoresis followed by immunoblotting using antisera from experimentally infected mice. Mass spectrometry analysis of the 32 immunoreactive protein spots resulted in the identification of 12 distinct proteins. Among them, 11 proteins were known antigens of Candida albicans, whereas Idh2p was identified for the first time as an immunogenic protein of Candida species. Recombinant Idh2p was expressed in Escherichia coli, and its antigenicity was verified by immunoblot analysis. CONCLUSIONS: An immunoproteomic approach was successfully applied to identify immunogenic proteins of C. parapsilosis, with Idh2p as a novel candidate antigen. The identified antigens may serve as potential biomarkers for development of diagnostic assay and/or vaccine for C. parapsilosis. SIGNIFICANCE AND IMPACT OF THE STUDY: This work represents the first immunoproteomic analysis of C. parapsilosis, which provides new insights into host-pathogen interactions and pathogenesis of C. parapsilosis. The immunogenic proteins could be studied as biomarker candidates for C. parapsilosis.

Candida albicans isolates from a Malaysian hospital exhibit more potent phospholipase and haemolysin activities than non-albicans Candida isolates.

This study was aimed at determining the phospholipase and haemolysin activity of Candida isolates in Malaysia. A total of 37 Candida clinical isolates representing seven species, Candida albicans (12), Candida tropicalis (8), Candida glabrata (4), Candida parapsilosis (1), Candida krusei (4), Candida orthopsilosis (1) and Candida rugosa (7) were tested. In vitro phospholipase activity was determined by using egg yolk plate assay whereas in vitro haemolysin activity was tested by using blood plate assay on sheep blood Sabouraud's dextrose agar (SDA) enriched with glucose. Phospholipase activity was detected in 75% (9 out of 12) of the C. albicans isolates. Among the 25 non- C. albicans Candida isolates, phospholipase activity was detected in only 24% of these isolates. The phospholipase activity of C. albicans was significantly higher than that of the non- C. albicans Candida isolates (P=0.002). Haemolysin activity was detected in 100% of the C. albicans, C. tropicalis, C. glabrata, C. krusei, C. parapsilosis, and C. orthopsilosis isolates while 75% of the C. krusei isolates and 12.3% of the C. rugosa isolates showed haemolysin activity. The haemolytic activity of C. albicans was significantly higher than that of the non- C. albicans Candida isolates (P=0.0001).The findings in this study indicate that C. albicans isolates in Malaysia may possess greater virulence potential than the non-albicans species.

Candida and invasive candidiasis: back to basics.

The ubiquitous Candida spp. is an opportunistic fungal pathogen which, despite treatment with antifungal drugs, can cause fatal bloodstream infections (BSIs) in immunocompromised and immunodeficient persons. Thus far, several major C. albicans virulence factors have been relatively well studied, including morphology switching and secreted degradative enzymes. However, the exact mechanism of Candida pathogenesis and the host response to invasion are still not well elucidated. The relatively recent discovery of the quorum-sensing molecule farnesol and the existence of quorum sensing as a basic regulatory phenomenon of the C. albicans population behavior has revolutionized Candida research. Through population density regulation, the quorum-sensing mechanism also controls the cellular morphology of a C. albicans population in response to environmental factors, thereby, effectively placing morphology switching downstream of quorum sensing. Thus, the quorum-sensing phenomenon has been hailed as the 'missing piece' of the pathogenicity puzzle. Here, we review what is known about Candida spp. as the etiological agents of invasive candidiasis and address our current understanding of the quorum-sensing phenomenon in relation to virulence in the host.

Identification of local clinical Candida isolates using CHROMagar Candida™ as a primary identification method for various Candida species.

The objective of our study was to study the effectiveness of CHROMagar Candida™ as the primary identification method for various clinical Candida isolates, other than the three suggested species by the manufacturer. We studied 34 clinical isolates which were isolated from patients in a local teaching hospital and 7 ATCC strains. These strains were first cultured in Sabouraud dextrose broth (SDB) for 36 hours at 35ºC, then on CHROMagar plates at 30ºC, 35ºC and 37ºC. The sensitivity of this agar to identify Candida albicans, Candida dubliniensis, Candida tropicalis, Candida glabrata, Candida rugosa, Candida krusei and Candida parapsilosis ranged between 25 and 100% at 30ºC, 14% and 100% at 35ºC, 56% and 100% at 37ºC. The specificity of this agar was 100% at 30ºC, between 97% and 100% at 35ºC, 92% and 100% at 37ºC. The efficiency of this agar ranged between 88 and 100% at 30ºC, 83% and 100% at 35ºC, 88% and 100% at 37ºC. Each species also gave rise to a variety of colony colours ranging from pink to green to blue of different colony characteristics. Therefore, the chromogenic agar was found to be useful in our study for identifying clinical Candida isolates.

Identification of local clinical Candida isolates using CHROMagar CandidaTM as a primary identification method for various Candida species

The objective of our study was to study the effectiveness of CHROMagar CandidaTM as the primary identification method for various clinical Candida isolates, other than the three suggested species by the manufacturer. We studied 34 clinical isolates which were isolated from patients in a local teaching hospital and 7 ATCC strains. These strains were first cultured in Sabouraud dextrose broth (SDB) for 36 hours at 35ºC, then on CHROMagar plates at 30ºC, 35ºC and 37ºC. The sensitivity of this agar to identify Candida albicans, Candida dubliniensis, Candida tropicalis, Candida glabrata, Candida rugosa, Candida krusei and Candida parapsilosis ranged between 25 and 100% at 30ºC, 14% and 100% at 35ºC, 56% and 100% at 37ºC. The specificity of this agar was 100% at 30ºC, between 97% and 100% at 35ºC, 92% and 100% at 37ºC. The efficiency of this agar ranged between 88 and 100% at 30ºC, 83% and 100% at 35ºC, 88% and 100% at 37ºC. Each species also gave rise to a variety of colony colours ranging from pink to green to blue of different colony characteristics. Therefore, the chromogenic agar was found to be useful in our study for identifying clinical Candida isolates

In vitro activity of fluconazole and voriconazole against clinical isolates of Candida spp. by E-test method.

The in vitro susceptibility of clinical Candida isolates towards fluconazole and voriconazole was determined using the E-test method. A total of 41 clinical isolates recovered from patients since 2004 until 2009 from two local hospitals in Kuala Lumpur, Malaysia were used. These comprised Candida tropicalis, Candida albicans, Candida krusei, Candida parapsilosis, Candida rugosa, Candida dubliniensis and Candida glabrata. Strains from American Type Culture Collection were used as quality control. Lawn cultures of the isolates on RPMI-1640 agar medium supplemented with 2% glucose were incubated with the E-test strips at 35ºC for 48 h. Our results show that 71% were susceptible to fluconazole and 90% were susceptible to voriconazole. All strains of C. krusei were resistant to fluconazole and 50% were susceptible in a dose-dependent manner to voriconazole. There were 66% and 33% of C. glabrata that were resistant to fluconazole and voriconazole. Our study revealed that majority of the clinical Candida isolates was susceptible to fluconazole and voriconazole with a small percentage being resistant to both the drugs.

High prevalence of human papillomavirus DNA detected in cervical swabs from women in southern Selangor, Malaysia.

Persistent high-risk human papillomavirus (HPV) infection is known to play an important role in the genesis of cervical cancer. Since new screening and prevention strategies, namely improved HPV testing and HPV vaccination have been aggressively promoted recently, it is crucial to investigate the HPV distribution in Malaysia in order to maximize their cost-effectiveness. This study was therefore conducted to assess the HPV type distribution in the most populous region, the state of Selangor. A total of 200 cervical swab samples were collected in two health-screening campaigns, and also from women attending obstetrics and gynecology clinics in several hospitals in Selangor. DNA extraction was performed and HPV DNA was detected via nested PCR using MY09/MY11 as outer primers and GP5+/GP6+ as inner primers which target the L1 gene of the viral genome. The purified PCR products were subjected to automated DNA sequencing to determine the HPV genotype. Out of 180 ß-globin positive samples, 84 (46.7%) were positive for HPV DNA. The most common HPV type found was high-risk oncogenic type 16 (40%), followed by HPV type 18 (3.3%), HPV 33 (1.7%), HPV 31 (0.6%), and low-risk HPV 87 (0.6%). Our study confirmed that nested PCR method is highly sensitive in detecting HPV DNA even in low risk patients. Since a relatively high prevalence rate of HPV infection was found in this population, prompt healthcare policy changes to bring about implementation of early HPV vaccination program is desirable to prevent a high incidence of cervical cancer.

Molecular identification of Candida orthopsilosis isolated from blood culture.

The incidence of candidemia and invasive candidiasis have increased markedly due to the increasing number of immunocompromised patients. There are five major medically important species of Candida with their frequency of isolation in the diminishing order namely Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata and Candida krusei. In addition, there are numerous other species of Candida which differ in their genetic makeup, virulence properties, drug susceptibilities and sugar assimilation capabilities. In this report, an unusual Candida species was isolated from the blood of two leukaemic patients. Conventional culture and biochemical tests identified the Candida species as C. parapsilosis. Using fungal-specific oligonucleotide primers ITS1 and ITS4, we managed to amplify the ribosomal RNA gene and its internal transcribed spacer region from the genomic DNA of these isolates. The PCR products were then purified and subjected to automated DNA sequencing using BLAST and CLUSTAL sequence analysis identified these isolates to be Candida orthopsilosis. Candida orthopsilosis is a new species recently identified in 2005, being morphologically indistinguishable from C. parapsilosis and was previously classified as a subspecies of C. parapsilosis. This report highlights the importance of complementing traditional culture and biochemical-based identification methods with DNA-based molecular assays such as PCR as the latter is more superior in terms of its discriminatory power and speed.

Inhibition of hyphae formation and SIR2 expression in Candida albicans treated with fresh Allium sativum (garlic) extract.

AIMS: The aims of the present study were to determine whether Allium sativum (garlic) extract has any effect on the morphology transformation of Candida albicans, and to investigate whether it could alter the gene expression level of SIR2, a morphogenetic control gene and SAP4, a gene encoding secreted aspartyl proteinase. METHODS AND RESULTS: Candida albicans cells were incubated with a range of concentrations of fresh garlic extract, and the morphology was monitored via light microscopy. Garlic extract treatment caused the transition of yeast form to hyphal form to be obviated. The expression of SIR2 was down-regulated from 1.2- to 2.5-fold with increasing concentration of the garlic extract, as determined from relative quantitative reverse transcription-polymerase chain reaction. There was no difference in the SAP4 expression in control vs treated cultures. CONCLUSIONS: Garlic and its bioactive components have the ability to suppress hyphae production and to affect the expression level of SIR2 gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Hyphal production is an essential virulence determinant of C. albicans for invasive infections, therefore garlic and its constituents can be effective not only against colonizing C. albicans strains present in mucosal infections, but also virulent strains causing systemic or invasive candidiasis.

The mycological and molecular study of Hortaea werneckii isolated from blood and splenic abscess.

Hortaea werneckii is an environmental dematiaceous fungus found in the halophilic environment. It causes tinea nigra. We report the isolation of H. werneckii from blood and splenic abscess of two patients with acute myelomonocytic leukaemia. H. werneckii grew at room temperature but not at 37 degrees C, it was identified by biochemical tests, growth characteristics and the presence of conspicuous collarette intercalary on dividing yeast cells. The use of specific oligonucleotide primer Hor-F (5'-TGGACACCTTCA TAACTCTTG-3') and Hor-R (5'-TCACAACGCTTAGAGACGG-3') confirmed the two isolates were H. werneckii. The sequence for 281 nucleotide of HW299 and HW403 were 99% identical but differed only in one nucleotide. In vitro anti-fungal susceptibility testing showed that the isolates were resistant to amphotericin B and flucytosine.