[Show abstract][Hide abstract] ABSTRACT: Background: Pythium insidiosum is the etiologic agent of pythiosis, a life-threatening infectious disease. Diagnosis of pythiosis is difficult and often delayed. Early diagnosis can lead to prompt treatment, and therefore a better prognosis for patients with pythiosis. Molecular diagnostic techniques are useful if microbiological and immunological assays are not available, or in cases of suspected pythiosis that test negative by other methods. So far, PCR identification of P. insidiosum has been largely relied on amplification of the rDNA region.
Objective: To evaluate the diagnostic performance of Dx3 and Dx4 primers specific for a putative gene for exo-1,3-β-glucanase (PinsEXO1), which encodes a specific immunogen of P. insidiosum, for rapid single-round PCR identification of P. insidiosum, in comparison with the previously-reported rDNA-specific primers, ITSpy1 and ITSpy2.
Materials and Methods: Genomic DNA (gDNA) from 35 P. insidiosum isolates and 48 control organisms were prepared to evaluate the diagnostic performance of the PinsEXO1- and rDNA-specific primers.
Results: When amplifying the control gDNA by using the Dx3/4 and ITSpy1/2 primer sets, no PCR product was observed, indicating that both primer sets had 100% detection specificity. When amplifying the P. insidiosum gDNA, the Dx3/4 primers provided an expected 550-bp amplicon for all 35 isolates, while the ITSpy1/2 primers provided an expected 230-bp amplicon for only 32 isolates. Thus, detection sensitivity of the Dx3/4 and
ITSpy1/2 primer sets were 100% and 91%, respectively.
Conclusion: By using the Dx3/4 primers, PinsEXO1 was an alternative, efficient, and novel PCR target for rapid single-round PCR identification of P. insidiosum.
Asian biomedicine 10/2014; 8(5):637-644. · 0.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: The fungus-like organism Pythium insidiosum is the causative agent of a life-threatening tropical infectious disease, pythiosis, which has high rates of morbidity and mortality. A lack of reliable diagnostic tools and effective treatments for pythiosis presents a major challenge to healthcare professionals. Unfortunately, surgical removal of infected organs remains the default treatment for pythiosis. P. insidiosum is an understudied organism. In-depth study of the pathogen at the molecular level could lead to better means of infection control. High quality genomic DNA (gDNA) is needed for molecular biology-based research and application development, such as: PCR-assisted diagnosis, population studies, phylogenetic analysis, and molecular genetics assays.
Objective: To evaluate quality and quantity of the P. insidiosum gDNA extracted by three separate protocols intended for fungal gDNA preparation.
Material and Method: Seven P. insidiosum isolates were subjected to gDNA extraction by using conventional-extraction, rapid-extraction, and salt-extraction protocols.
Results: The conventional protocol offered the best gDNA in terms of quality and quantity, and could be scaled up. The rapid-extraction protocol had a short turnaround time, but the quality and quantity of the gDNA obtained were limited. The salt-extraction protocol was simple, rapid, and efficient, making it appealing for high throughput preparation of small-scale gDNA samples.
Conclusion: Compared to rapid-extraction protocol, both conventional-extraction and salt-extraction protocols provided a better quality and quantity of gDNA, suitable for molecular studies of P. insidiosum. In contrast to the other two methods, the salt-extraction protocol does not require the use of hazardous and expensive materials such as phenol, chloroform, or liquid nitrogen.
Journal of the Medical Association of Thailand = Chotmaihet thangphaet 03/2014; 97(3):342-8.
[Show abstract][Hide abstract] ABSTRACT: Describe the clinical characteristics, treatment, outcomes, complications, and factors associated with mortality of cryptococcosis in HIV-negative patients.
A retrospective cohort study was conducted among HIV-negative adult patients with positive culture for Cryptococcus neoformans between 2005 and 2010.
Forty-nine HIV-negative patients were identified with median (IQR) age of 62.5 (45.5-71.9) years of which 40.8% were male. The common underlying medical conditions were cardiovascular diseases (36.7%). The common sites of positive culture were cerebrospinal fluid/intracerebral abscess (46.9%), blood (36%), and sputum/bronchoalveolar larvage fluid (28.6%). Twenty-nine (59.2%) patients had co-infections with another organism, such as Gram-negative bacteria (24.4%), M. tuberculosis (17.8%), and Gram-positive bacteria (13.3%). The common clinical presentations were fever (67.3%), alteration of consciousness (34.7%), and headache (26.5%). Complication was detected in 61.2% such as acute kidney injury (47.0%), coma (38.8%), and shock (22.4%). The overall mortality was 51%. By multivariate logistic regression, factors associated with mortality were alteration of consciousness (adjusted OR = 6.85; 95% CI: 1.41-33.28, p = 0.017) and co-infections (adjusted OR = 5.32; 95% CI: 1.25-22.69, p = 0.024).
The mortality rate of HIV-negative patients with cryptococcosis is very high. Early recognition and treatment of cryptococcosis in HIV-negative patients are crucial and may improve the outcome.
Journal of the Medical Association of Thailand = Chotmaihet thangphaet 01/2014; 97(1):36-43.
[Show abstract][Hide abstract] ABSTRACT: Cerebral mycosis is a significant cause of morbidity among immunocompromised populations. We present here a case of cerebral infection with Scedosporium apiospermum and Phaeoacremonium parasiticum in a 49-year-old renal transplant recipient. Fourteen years after renal transplantation, the patient presented with invasive pulmonary aspergillosis treated with intravenous liposomal amphotericin B. The patient had clinical and radiographic improvement. However, 6 weeks later, the patient presented with cerebral infection. Magnetic resonance imaging revealed multiple rim enhancing brain abscesses. Brain and cerebrospinal fluid cultures ultimately grew Scedosporium apiospermum and Phaeoacremonium parasiticum. The patient was treated with voriconazole for 6 months and had clinical and radiologic improvement. We believe this is the first reported case of co-infection of the brain with scedosporiosis and phaeohyphomycosis in a renal transplant recipient, who had received intravenous liposomal amphotericin B. Voriconazole may represent a new therapeutic option for these simultaneous infections in the brain.
The Southeast Asian journal of tropical medicine and public health 05/2013; 44(3):484-9. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Clostridium difficile poses as the most common etiologic agent of nosocomial diarrhea. Although there are many diagnostic methods to detect C. difficile directly from stool samples, the nucleic acid-based approach has been largely performed in several laboratories due to its high sensitivity and specificity as well as rapid turnaround time. In this study, a multiplex PCR was newly designed with recent accumulated nucleotide sequences. The PCR testing with various C. difficile ribotypes, other Clostridium spp., and non-Clostridium strains revealed 100% specificity with the ability to detect as low as ~22 genomic copy number per PCR reaction. Different combinations of sample processing were evaluated prior to multiplex PCR for the detection of C. difficile in fecal samples from hospitalized patients. The most optimal condition was the non-selective enrichment at 37°C for 1 h in brain heart infusion broth supplemented with taurocholate, followed by the multiplex PCR. The detection limit after sample processing was shown as being 5 spores per gram of fecal sample. Two hundred and thirty-eight fecal samples collected from the University affiliated hospital were analyzed by the enrichment multiplex PCR procedure. The results suggested that the combination of sample processing with the high-performance detection method would be applicable for routine diagnostic use in clinical setting.
BioMed research international. 01/2013; 2013:875437.
[Show abstract][Hide abstract] ABSTRACT: Few studies have described the pattern of bloodstream infections (BSI) among HIV-infected patients in the highly active antiretroviral therapy (HAART) era, particularly in resource-limited settings. A retrospective cohort study was conducted among 140 HIV-infected patients who had a positive blood culture from 2004-2008. Of the 140 patients, 91 (65%) were male with a mean (SD) age of 38 (9.1) years and a median (IQR) CD4 cell count of 32 (9-112) cells/mm(3). Community-acquired infection was detected in 89% of patients. The blood cultures contained Gram-negative bacteria, 40%; fungi, 24%; Mycobacterium spp., 20%; and Gram-positive bacteria, 16%. Common causative pathogens were Cryptococcus neoformans, 21%; Salmonella spp., 15%; and Mycobacterium tuberculosis, 12%. Common focal sites of infection were the central nervous system, 24%; respiratory tract, 20%; and gastrointestinal tract, 18%. CD4 cell count (OR, 0.61 per 50 cells/mm(3) increment; 95% CI, 0.39-0.96; P = 0.031) was the only factor associated with mycobacterial or fungal BSI. The crude mortality was 21%. HAART (OR, 0.23; 95% CI, 0.01-0.77; P = 0.017), focal infection (OR, 0.31; 95% CI, 0.10-0.97; P = 0.044), and complication (e.g., shock) (OR, 9.26; 95% CI, 3.25-26.42; P < 0.001) were the predictive factors of mortality. In conclusion, opportunistic infections are still the leading causes of BSI among HIV-infected patients in the HAART era.
Japanese journal of infectious diseases. 01/2012; 65(1):28-32.
[Show abstract][Hide abstract] ABSTRACT: Determine and compare the clinicopathological findings of cerebral aspergillosis with cerebral candidiasis.
The medical records with cerebral aspergillosis and cerebral candidiasis in Ramathibodi Hospital between January 1997 and December 2008 were analyzed. The criterion for the diagnosis of cerebral aspergillosis and cerebral candidiasis was the evidence of fungal elements from histopathologic section. The age, gender neurological manifestations, duration of symptom, associated underlying disease, predisposing risk factor, laboratory data, extent of systemic organ involvement and treatment outcome were analyzed.
The present study included cerebral aspergillosis (n = 41) and candidiasis (n = 15). There were 23 male and 33 female patients. The mean and median ages at diagnosis were 39.7 and 45 years, respectively (range, 1 month to 87 years). The clinical presentations included alteration of consciousness (69.6%), fever (60.7%), weakness of the extremity (14.3%), cranial nerve palsy (12.5%), headache (12.59%) and seizure (5.4%). One third of the cases had underlying hematologic malignancy. The cerebral aspergillosis and cerebral candidiasis were associated with corticosteroids treatment in 32.1%. The frequent associated sites of fungal infection included the lungs (73.2%), alimentary tract (33.9%) and sinonasal tract (19.6%).
A diagnosis of cerebral aspergillosis and cerebral candidiasis requires a high index of suspicion especially in immunocompromised patients who presented with alteration of consciousness, fever, focal neurological deficit, headache, and seizure. The patients with cerebral aspergillosis and cerebral candidiasis manifest with similar clinicopathologic features. However, the sinonasal tract infection and abscess formation are more common in cerebral aspergillosis. Associated alimentary tract infection is commonly seen in cerebral candidiasis.
Journal of the Medical Association of Thailand = Chotmaihet thangphaet 12/2010; 93(12):1443-50.
[Show abstract][Hide abstract] ABSTRACT: Pythium insidiosum causes a potentially life-threatening infectious disease called pythiosis. An early, accurate diagnosis is important, since prompt treatment leads to a better prognosis. Unsuccessful attempts to isolate the organism have been associated with specimens subjected to lower temperatures. We analyzed growth of P. insidiosum at various temperatures. Culture at low (8 degrees C) and high (42 degrees C) temperatures resulted in death or inhibited growth of the organism. Culture under optimal temperatures (28 and 32 degrees C) was important for successful isolation of P. insidiosum.
The Southeast Asian journal of tropical medicine and public health 11/2010; 41(6):1462-6. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A total of 83 Acinetobacter baumannii isolates from patients attending a tertiary care university hospital in Thailand were investigated for their clonal relatedness, antimicrobial susceptibility profiles, and integron carriage. Susceptibility profiles showed that 56 (67%) of these isolates exhibited multiple drug resistance (MDR). Pulsed-field gel electrophoresis (PFGE) showed that 73% of these resistant isolates were clustered into three predominant PFGE types: 6, 7, and 36. This suggested that the high number of isolates exhibiting MDR phenotypes observed in the hospital is, to some extent, due to the spread of these three resistant clones. Class 1 integrase genes were detected in all MDR isolates belonging to PFGE type 6, most MDR isolates belonging to PFGE type 7 and none of the isolates belonging to PFGE type 36. Five different class 1 gene cassette arrays, dfrA1-orfC, bla(IMP-14)-aac6', aacA4- catB8-aadA1, aacC1-orfX-orfX'-aadA1a, and aacC1-orfX-orfX-orfX'-aadA1a, were identified, of which the bla(IMP-14)-aac6' array has only been found in Thai isolates. Two isolates identified in this study carried a class 2 integrase gene with a 2.2 kb cassette array containing aadA1-sat-dfrA1.
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to determine the clinicopathologic findings of invasive and non-invasive fungal rhinosinusitis and to compare the features of the two diseases. The medical records of patients with invasive and noninvasive fungal rhinosinusitis at Ramathibodi Hospital between July 1999 and June 2009 were analyzed. The criterion for the diagnosis of fungal rhinosinusitis was the evidence of fungal elements from histopathologic section on sinonasal specimens. The age, gender, clinical manifestations, duration of symptoms, associated diseases, laboratory data, results of mycotic culture and treatment outcomes were analyzed. The relationship between fungal rhinosinusitis and patient characteristics as well as clinical presentations were assessed. The fungus-attributable mortality rate was determined. The study included 43 cases of invasive fungal rhinosinusitis and 68 cases of non-invasive fungal rhinosinusitis. There were 44 male, and 67 female patients. The mean age at diagnosis was 54.6 years (range: 5 to 86 years). A total of 70 (63.1%) were attributed to aspergillosis, 8 (7.2%) to candidiasis, 6 (5.4%) to zygomycosis, 4 (3.6%) to phaeohyphomycosis, 1 (0.9%) to pseudallescheriasis, 1 (0.9%) to entomophthoromycosis and 21 (18.9%) to nonspecific fungi. Cultures from sinonasal tissues were positive for fungus in 37 of 87 cases (42.5%). The clinical presentations of fungal rhinosinusitis included nasal stuffiness (27.9%), nasal discharge (27.9%), facial pain (27.9%), fever (24.3%) and headache (19.8%). One-fifth of cases had an underlying hematologic malignancy. Invasive fungal rhinosinusitis was significantly associated with hematologic malignancy and neutropenia. Fungus-attributable mortality rate was 44.2% in invasive fungal rhinosinusitis. Early antifungal therapy and surgical drainage were associated with a survival advantage.
The Southeast Asian journal of tropical medicine and public health 03/2010; 41(2):442-9. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to report a case of contact lens-related Pythium insidiosum corneal ulcer.
The authors conducted an interventional case report. We report the clinical presentations, histopathologic findings, and treatments of a patient with P. insidiosum keratitis associated with contact lens wear.
A 22-year-old Thai woman presented to our clinic with a nonresponsive, progressive corneal ulcer of the left eye. Slit lamp examination showed a large central ulcer measuring 5.4 x 5.2 mm with underlying dense stromal infiltrates surrounded by subepithelial and superficial stromal opacity in a reticular pattern along with radial perineural-like infiltrates. Histopathologic examination revealed broad, branched, thin-walled nonparallel hyaline hyphae with rare septates consistent with P. insidiosum. The corneal culture confirmed the diagnosis. The infection recurred after multiple therapeutic penetrating keratoplasties. Enucleation was eventually performed to eradicate the infection. No definite report of contact lens-related P. insidiosum keratitis existed in the literature.
Pythium keratitis may present with central ulcer and radial keratoneuritis similar to acanthamoeba keratitis. Contact lens wear can be a risk factor for this infection. Increasing awareness, early diagnosis, and intervention may improve the prognosis.
[Show abstract][Hide abstract] ABSTRACT: Floods are natural disasters that occur occasionally in Thailand. The most common form skin diseases due to floods are infectious dermatoses especially superficial fungal infection. However the microbiologic evidences have not been evaluated.
To evaluate the most common skin diseases during floods and identify the organism that causes skin maceration at web space(s) of toes (Hong Kong foot).
Patients who complained of skin problems were evaluated at the temporary outpatient clinic during October 2006. Skin specimens from all patients who had itches and skin maceration at web space(s) of toes were cultured.
Ninety-six patients were evaluated (38 males and 58 females). Eczema was the most prevalent dermatosis, which accounted for 34.5% of the total skin problems and the great majority of these cases were irritant contact dermatitis. Sixteen cases presented with itch and skin maceration at web space(s) of toes. All of them were colonized with various microorganisms. Gram-negative bacilli were the most prevalent ones and were found in 14 out of 16 specimens. Fungal culture was positive in only two specimens.
Eczema is the most common dermatosis during floods. Skin maceration at web space(s) of toes, which were thought to be fungal infection, are chronic irritant dermatitis with secondary bacterial colonization. Only a few cases were fungal infection. Microbiologic investigation should be done in these patients. Unfortunately, it is not practical in such a situation. Topical medications that have the combination of antiinflammatory, antibacterial and antifungal properties are the most suitable medications.
Journal of the Medical Association of Thailand = Chotmaihet thangphaet 05/2008; 91(4):479-84.
[Show abstract][Hide abstract] ABSTRACT: Determine the clinicopathological findings in autopsy cases with invasive fungal infection.
The autopsy and medical records with invasive fungal infection in Ramathibodi Hospital between January 1997 and December 2006 were analyzed. The criterions for the diagnosis of invasive fungal infection were the evidence of fungal elements from histopathological section. The age, gender underlying predisposing risk factors for the disease, clinical manifestations, extent of systemic organ involvement documented morphologically at autopsy, and fungal culture were analyzed
There were 155 autopsy cases (73 male, 82 female; mean age 45.3 years, range 3 months to 87 years) with the diagnosis of invasive fungal infection. The common clinical presentations were fever (55.5%), and dyspnea (26.5%). The invasive fungal infection was associated with hematologic malignancy in 31%. The common mycoses were aspergillosis and candidiasis, which were observed in 88 and 80 cases, respectively. There were 32 cases (20.6%) of mixed fungal infection. Cultures from autopsy materials were positive for fungus in 80 cases out of 99 cases (80.8%). The most frequent site of fungal infection was in the lungs (74.8%), followed by gastrointestinal tract (28.4%), and brain (26.5%). Invasive fungal infection was diagnosed intravitally in 63.9% of total cases.
A diagnosis of invasive fungal infection requires a high index of suspicion, especially in immunocompromised patients who presented with prolonged fever Clinical specimens must be sent for histopathology and fungal culture for a definite diagnosis and an appropriate management. Therefore, the physician should inform the laboratory if invasive fungal infection is suspected because special media are necessary for the best recovery of fungi. In addition, the present study underscores the significance of autopsy as a diagnostic method and means of medical quality control.
Journal of the Medical Association of Thailand = Chotmaihet thangphaet 01/2008; 90(12):2630-7.
[Show abstract][Hide abstract] ABSTRACT: The oomycetous, fungus-like, aquatic organism Pythium insidiosum is the etiologic agent of pythiosis, a life-threatening infectious disease of humans and animals that has been increasingly reported from tropical, subtropical, and temperate countries. Human pythiosis is endemic in Thailand, and most patients present with arteritis, leading to limb amputation and/or death, or cornea ulcer, leading to enucleation. Diagnosis of pythiosis is time-consuming and difficult. Radical surgery is the main treatment for pythiosis because conventional antifungal drugs are ineffective. The aims of this study were to evaluate the use of Western blotting for diagnosis of human pythiosis, to identify specific immunodominant antigens of P. insidiosum, and to increase understanding of humoral immune responses against the pathogen. We performed Western blot analysis on 16 P. insidiosum isolates using 12 pythiosis serum samples. These specimens were derived from human patients with pythiosis who had different forms of infection and lived in different geographic areas throughout Thailand. We have identified a 74-kDa immunodominant antigen in all P. insidiosum isolates tested. The 74-kDa antigen was also recognized by sera from all patients with pythiosis but not by control sera from healthy individuals, patients with thalassemia, and patients with various infectious diseases, indicating that Western blot analysis could facilitate diagnosis of pythiosis. Therefore, the 74-kDa antigen is a potential target for developing rapid serodiagnostic tests as well as a therapeutic vaccine for pythiosis. These advances could lead to early diagnosis and effective treatment, crucial factors for better prognosis for patients with pythiosis.
Journal of Clinical Microbiology 06/2006; 44(5):1674-80. · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human pythiosis is an emerging, fatal, infectious disease caused by Pythium insidiosum and occurs in both tropical and subtropical countries. Thalassemic patients, farmers, and aquatic-habitat residents are predisposed to this disease. Delayed treatment due to the long time required for isolation and identification of the causative organism, as well as the difficulty in obtaining internal organ specimens, results in high morbidity and mortality. To facilitate rapid diagnosis, an in-house enzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin G antibodies against P. insidiosum was developed and evaluated for the diagnosis and monitoring of human pythiosis. Sixteen sera were collected from seven culture-proven human pythiosis cases. A total of 142 sera from thalassemic patients, from patients with other infectious diseases, and from healthy blood donors served as controls. All sera were tested in duplicate. By choosing a suitable cutoff point to maximize sensitivity and specificity, sera from pythiosis cases were all determined to be positive, whereas sera from control groups were all determined to be negative. ELISA signals from serial samples of sera taken from treated patients showed gradually declining levels of antibodies to P. insidiosum. The ELISA test was highly sensitive (100%) and specific (100%) and was useful for early diagnosis and for monitoring the treatment for pythiosis.