Identification of <Emphasis Type="Italic">Penicillium marneffei</Emphasis> in Paraffin-Embedded Tissue Using Nested PCR

Penicillium marneffei is one of the unique thermally dimorphic fungi in Penicillium species that causes a disseminated, progressive and life threatening infection in immunocompromised patients. The diagnosis of Penicilliosis marneffei depends on culture that may delay the treatment due to the time-consuming process. In the present study, we evaluated the specificity and sensitivity of nested PCR to identify Penicillium marneffei from paraffin-embedded tissue. Two sets of oligonucleotide primers were derived from the sequence of 18S rRNA of Penicillium marneffei. The outer primers (RRF1 and RRH1) were specific to fungi. The inner primers (Pm1 and Pm2) were specific to Penicillium marneffei. The specific fragment of approximately 400 bp was amplified from all paraffin-embedded tissues from 14 patients with Penicilliosis marneffei and 10 bamboo rats. The detectable DNA concentration of single PCR and nested PCR were 14 pg/μl and 14 fg/μl, respectively. Further studies are required in order to use nested PCR for early diagnosis of the disease.     

Indigenous Case of Disseminated Histoplasmosis from the <Emphasis Type="Italic">Penicillium marneffei</Emphasis> Endemic Area of China

This is the first indigenous case of disseminated histoplasmosis reported from the Penicillium marneffei endemic area in southern China. It was diagnosed by histopathology of tissue, gross and microscopic morphology of the culture and PCR assay of the isolated fungus. Successful antifungal treatment was with itraconazole 400 mg/day for 5 months. This case suggests that histoplasmosis should be an important differential diagnosis in immunocompromised patients in southern China and South East Asia (the only endemic area for P. marneffei).     

Characteristics and Prognosis of Talaromyces marneffei Infection in Non-HIV-Infected Children in Southern China

Knowledge about the clinical and laboratory characteristics and prognosis of Talaromyces marneffei infection in children is limited. A retrospective study was conducted on pediatric patients with disseminated T. marneffei infection in a clinical setting. Extracted data included demographic information (age and sex), clinical features, laboratory findings, treatment, and prognosis. Eleven HIV-negative children were enrolled. The male/female ratio was 8:3. The median age of onset was 17.5 months (3.5–84 months). The mortality rate in these children was 36.36% (4/11). Seven children had underlying diseases. All of the children had multiple immunoglobulin abnormalities and immune cell decline. Ten children received voriconazole treatment, and most of the children (7/10) had a complete response to therapy at primary and long-term follow-up assessment; only three children died of talaromycosis. One patient recovered from talaromycosis but died of leukemia. The child who received itraconazole treatment also showed clinical improvement. No adverse events associated with antifungal therapies were recorded during and after the treatment. Talaromycosis is an indicator disease for undiagnosed severe immunodeficiencies in children. Awareness of mycoses in children by pediatricians may prompt diagnosis and timely treatment. Voriconazole is an effective, well-tolerated therapeutic option for disseminated T. marneffei infection in non-HIV-infected children.

     

Administration of Voriconazole in Disseminated <Emphasis Type="Italic">Talaromyces</Emphasis> (<Emphasis Type="Italic">Penicillium</Emphasis>) <Emphasis Type="Italic">Marneffei Infection</Emphasis>: A Retrospective Study

Talaromyces (Penicillium) marneffei infection is a fatal disseminated mycosis caused by the dimorphic fungus Talaromyces marneffei; the therapeutic strategies for this infectious disease are limited. The aim of this retrospective study was to evaluate the efficacy and safety of voriconazole for treating patients with disseminated T. marneffei infection with or without HIV infection in a clinical setting. Patients who intravenously received voriconazole (6 mg/kg q12 h for the first 24 h followed by 4 mg/kg q12 h) as the initial antifungal treatment were enrolled. The duration of the following antifungal treatment varied at the discretion of the investigators according to the patient responses. The primary global response was evaluated at Week 16 or at the end of treatment (EOT). Follow-up evaluations were performed at 6 months and 1 year after the EOT. Seventeen patients were enrolled in this study, but three were not evaluable because the treatment was prematurely discontinued. Among the remaining fourteen patients, ten patients had complete response and three had partial response at Week 16. Only one patient was determined to have failed response. Follow-up assessments in eleven patients showed that eight patients were cured and the remaining three patients relapsed at 6 months after the EOT. These eight patients were assessed 1 year later, and none of them had relapsed. No adverse events associated with voriconazole were recorded during the treatment. The results from our study suggest that voriconazole is an effective, well-tolerated therapeutic option for disseminated T. marneffei infection.     

Characteristics and Prognosis of Talaromyces marneffei Infection in HIV-positive Children in Southern China

Knowledge about the clinical characteristics and prognostic factors of Talaromyces marneffei infection in children is limited, especially in HIV-positive children. We performed a retrospective study of all HIV-positive pediatric inpatients with T. marneffei infection in a tertiary hospital in Southern China between 2014 and 2019 and analyzed the related risk factors of poor prognosis using logistic regression. Overall, 28 cases were enrolled and the prevalence of talaromycosis in AIDS children was 15.3% (28/183). The median age of the onset was 8 years (range: 1–14 years). The typical manifestation of skin lesion with central umbilication was not common (21.4%). All the children had very low CD4⁺ cell counts (median 13.5 cells/μL, range: 3–137 cells/μL) on admission. 92.9% children were misdiagnosed and talaromycosis was only noted after positivity for HIV infection. 89.3% diagnoses of T. marneffei infections were based on positive blood cultures, with a long culture time (median 7 days, range from 3–14 days). The sensitivity of fungus 1,3-β-D-glucan assay was 63.2%. Amphotericin B was superior to itraconazole in the induction antifungal therapy of talaromycosis in HIV-positive children. A six-month follow-up revealed a 28.6% mortality. Lower ratio of CD4⁺/CD8⁺ and amphotericin B treatment not over 7 days predicted poor prognosis. Our retrospective study provided an overview and update on the current knowledge of talaromycosis in HIV-positive children. Pediatricians in endemic areas should be aware of mycoses to prevent misdiagnosis. 1,3-β-D-glucan assay did not show optimal sensitivity. Amphotericin B treatment over 7 days can improve poor prognosis.

     

Acute Disseminated <Emphasis Type="Italic">Talaromyces marneffei</Emphasis> in An Immunocompetent Patient

Talaromyces marneffei (also called Penicilliosis Marneffei or T. marneffei) is a rare fungal disease that is prevalent mainly in Southeast Asia and commonly seen in immunocompromised hosts. It was rarely observed in immunocompetent hosts. We report a case of acute disseminated T. marneffei in an immunocompetent patient in the non-prevalent region. This patient had never visited the endemic area. The patient experienced a persistent fever. Brain CT and magnetic resonance imaging (MRI) showed a mass in the right frontal with osteolytic damage. Excessive white blood cell (WBC) count and C-reactive protein content were observed. Antibiotics including meropenem and linezolid could not play an effect, and another two hard masses appeared in his right neck and front chest wall. The aspirates from the right frontal mass and bone marrow were cultured. The final diagnose of this infection was disseminated T. marneffei. After voriconazole treatment, all symptoms improved gradually. We present this case and aim to promote more clinicians and microbiologists in the non-endemic region to recognize this rare disease.     

A Case of Primary Localized Cutaneous Infection Due to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

Fusarium is a ubiquitous hyalohyphomycete isolated from food, widespread in the environment (plants, soil) and present at all latitudes. Fusarium oxysporum and Fusarium solani are the most frequent pathogenic species, followed by F. moniliforme and F. chlamydosporum. Infections due to this mold may be disseminated or localized. Localized forms include cutaneous and subcutaneous infection, onychomycosis, endophtalmitis, otitis, sinusitis, arthritis, osteomyelitis, and brain abscess. Disseminated forms are those in which two or more noncontiguous sites may be involved. These latter are observed in patients with severe neutropenia. Wounds, digital ulcers, onychomycosis, and paronychia are the typical cutaneous portal of entry. We report a case of primary localized cutaneous infection due to Fusarium in a 29-year-old woman presenting with a nodular lesion, partially ulcerated, asymptomatic on the first finger of the left hand, appeared 4 months earlier. Histological examination showed spongiosis and acanthosis in the stratum corneum, ulceration and inflammation with prevalently mononucleate cells and septate and branched fungal structures in the epidermis and in dermis. The fungus was identified as Fusarium oxysporum by culture of biopsy fragments on Sabouraud dextrose agar with chloramphenicol. The culture was deposited in the culture collection of the mycology section of IHEM, Brussels (IHEM21984 col no. 125). The patient had normal immune status and was successfully treated with surgical excision. Recovery was confirmed at follow-up 8 months later.     

Caenorhabditis elegans: A Simple Nematode Infection Model for Penicillium marneffei

Penicillium marneffei, one of the most important thermal dimorphic fungi, is a severe threat to the life of immunocompromised patients. However, the pathogenic mechanisms of P. marneffei remain largely unknown. In this work, we developed a model host by using nematode Caenorhabditis elegans to investigate the virulence of P. marneffei. Using two P. marneffei clinical isolate strains 570 and 486, we revealed that in both liquid and solid media, the ingestion of live P. marneffei was lethal to C. elegans (P<0.001). Meanwhile, our results showed that the strain 570, which can produce red pigment, had stronger pathogenicity in C. elegans than the strain 486, which can’t produce red pigment (P<0.001). Microscopy showed the formation of red pigment and hyphae within C. elegans after incubation with P. marneffei for 4 h, which are supposed to be two contributors in nematodes killing. In addition, we used C. elegans as an in vivo model to evaluate different antifungal agents against P. marneffei, and found that antifungal agents including amphotericin B, terbinafine, fluconazole, itraconazole and voriconazole successfully prolonged the survival of nematodesinfected by P. marneffei. Overall, this alternative model host can provide us an easy tool to study the virulence of P. marneffei and screen antifungal agents.     

Penicillium Marneffei N. Sp., Agent D'une Mycose Du Système Réticulo-Endothélial

Résumé 1) Une espèce nouvelle dePenicillium, P. marneffei,Segretain, Capponi, Sureau, a été isolée d'un rongeur du Centre Viet Nam,Rhizomys sinensis ou rat de bambou, provoquant la mortalité de cet animal en captivité,2) Ce champignon est expérimentalement pathogène pour la souris le hamster et le rat, atteints d'une réticulose macrophagique semblable à l'histoplasmose ou à la leishmaniose.3) Le champignon se développe dans les cellules réticulaires où il se présente sous forme ronde, ovale ou légèrement allongée. Il se multiplie par cloisonnement transversal.4)En culture à 37°,P. marneffei donne des cultures glabres, lisses ou plissées, de consistance crémeuse, ressemblant à une culture bactérienne et formées d'arthrospores.5)Par ses caractères botaniques, le champignon doit être considéré comme une espèce nouvelle, à rapprocher deP. janthinellum et deP.citrinum.

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Summary 1) A new species ofPenicillium, P. marneffei Segretain, Capponi, Sureau, has been isolated from a rodent of the center of Viet Nam,Rhizomys sinensis or bamboo rat; it causes the death of this animal living in captivity.2) This fungus is experimentally pathogen for mice, hamsters and rats: it produces a reticulosis similar to histoplasmosis or leishmaniosis.3) The fungus grows in the reticulo-endothelial cells in which its form is round, oval or slightly elongated. It multiplies by scissiparity.4) At 37° C the colony ofP. marneffei is glabrous, smooth or folded, resembling that of Bacteria and formed by arthrospores.5) By its botanical caracters, the fungus is considered as a new species to be classed nearP. janthinellum andP. citrinum.

     

Fatal <Emphasis Type="Italic">Talaromyces marneffei</Emphasis> Infection in a Patient with Autoimmune Hepatitis

Talaromyces marneffei, previously known as Penicillium marneffei, is the most important pathogenic thermally dimorphic fungus causing systemic mycosis in Southeast Asia. Traditionally, T. marneffei infection in human was mainly associated with acquired immunodeficiency syndrome caused by HIV infection. In recent years, there has been an increasing number of T. marneffei infections reported in non-HIV-infected patients with other immunocompromised conditions, including autoantibodies against interferon-gamma, systemic lupus erythematosis, solid organ transplantation, Job’s syndrome, hematological malignancies, and use of novel targeted therapies. In this article, we describe the first case of fatal T. marneffei infection in a patient with underlying autoimmune hepatitis, presented as fever without localizing features. The diagnosis of talaromycosis was confirmed with the identification of the fungi isolated from the blood culture specimen by conventional methods and using matrix-assisted laser desorption–ionization time-of-flight mass spectrometer. This case shows the importance of a high index of suspicion, particularly for such a highly fatal but potentially treatable fungal infection.     

Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the fungus Penicillium marneffei

Penicillium marneffei is an opportunistic fungal pathogen of humans, causing respiratory, skin, and systemic mycosis in south-east Asia. Here we describe the transformation of P. marneffei with Agrobacterium tumefaciens, and the optimization of the transformation procedure. Transformations in different combinations between A. tumefaciens stains (LBA4404 and EHA105) and binary vectors (pCB309A, pBI129A, and pCaMBIA1312A) showed that EHA105/pBI129A were the most efficient partners. Southern blot analysis suggested that 87.5 % of transformants obtained with this protocol displayed single hybridization bands, indicating a single insert of T-DNA in each of the transformants. Unique hybridization patterns, along with thermal asymmetric interlaced PCR (TAIL-PCR) analysis of T-DNA insertion sites, suggested that A. tumefaciens-mediated transformation may be a powerful tool for insertional mutagenesis in P. marneffei. Several mutants with altered phenotypes were obtained during the construction of the mutant library, indicating the usefulness of the approach for functional genetic analysis in this important fungal pathogen.     

Histoplasmosis: cytodiagnosis and review of literature with special emphasis on differential diagnosis on cytomorphology

N. Gupta, S. K. Arora, A. Rajwanshi, R. Nijhawan and R. Srinivasan
Histoplasmosis: cytodiagnosis and review of literature with special emphasis on differential diagnosis on cytomorphology Background: Human infection with Histoplasma capsulatum runs the gamut from asymptomatic to disseminated disease. In immunocompromised patients, a tiny inoculum can lead to widespread disseminated infection. Early diagnosis and initiation of treatment is therefore important. Objective: To review the cases of histoplasmosis diagnosed on fine needle aspiration cytology (FNAC) and to discuss the clinical presentation, associated inflammatory response, load of organisms and differential diagnosis on cytomorphology in these cases. Methods: Retrospective review of seven cases of histoplasmosis at a tertiary‐care centre during the period from 1998 to 2009 was performed. Clinical presentation along with cytomorphological features were studied and discussed in detail. Results: The mean age of patients was 48.6 years and six out of seven were male. History of immunodeficiency (HIV) was available in five cases. Six patients presented with peripheral and/or abdominal lymphadenopathy. One patient had nodular shadows in both lungs and two also had skin lesions. On cytological smears, a variable load of uniform round to oval, about 2–4 μm in diameter, budding yeasts were seen intracellularly (within histiocytes) as well as extracellularly. In one case (HIV positive), these organisms were also seen within neutrophil polymorphonuclear leucocytes. In two cases, an inflammatory response in the form of epithelioid cell granulomas along with multinucleated giant cells was seen. Conclusions: FNAC is a reliable tool to recognize infection with H. capsulatum in tissues. This infection can cause a variable inflammatory response, which should be considered while reporting on such cases.     

In Vitro Interactions of Calcineurin Inhibitors with Conventional Antifungal Agents Against the Yeast Form of Penicillium marneffei

Penicillium marneffei can cause a life-threatening disseminated mycosis in immunocompromised hosts. However, therapeutic strategies for the treatment of this infectious disease are limited. Reports of other fungi suggest that calcineurin inhibitors interact with antifungal agents to improve the treatment outcomes. Here, we evaluated the in vitro interaction of the calcineurin inhibitors cyclosporine A and tacrolimus (FK506) combined with conventional antifungal agents against the pathogenic yeast form of P. marneffei. We demonstrate that the combination of cyclosporine A with amphotericin B, itraconazole, or fluconazole was synergistic for 85, 65, and 30 % of P. marneffei strains, respectively. In contrast, no synergism was observed in all the combinations containing tacrolimus. Furthermore, antagonism was not observed for any combination. In conclusion, the therapeutic potential of a combinatory approach using the calcineurin inhibitor cyclosporine A with conventional antifungal drugs may lead to improved treatment regimens for P. marneffei infections. We propose that mechanism of action studies with cyclosporine A and antifungal agents is needed.     

Isolation of <Emphasis Type="Italic">Penicillium marneffei</Emphasis> From Soil and Wild Rodents in Guangdong,SE China

Infection by Penicillium marneffei is an important emerging public health problem, especially among travelers and inhabitants in SE China and SE Asia infected with human immunodeficiency virus (HIV). In recent years, the number of patients with penicilliosis marneffei (PM) has increased rapidly in Guangdong province, SE China. However, the natural habitat and transmission mode of the etiologic agent remains unclear. In this study, wild rats (Microtus, focus Rattus and Rhizomys pruinosus) and soil samples were collected from rat burrows, populated and rural areas from November 2007 to December 2008 for fungus cultures. All isolates, suspected of being P. marneffei, were identified by gross and microscopic morphology and ITS analysis. Sixteen of 23 (about 70%) bamboo rats were P. marneffei positive, whereas none was recovered from hamsters, loirs or soil. This suggests that as of today the bamboo rat is the exclusive natural reservoir for P. marneffei. Definite evidence that rodents are a part of the infectious cycle is still lacking.     

Study on the Clinical Features and Prognosis of <Emphasis Type="Italic">Penicilliosis marneffei</Emphasis> Without Human Immunodeficiency Virus Infection

Objective

To improve the diagnosis and treatment of Penicilliosis marneffei without human immunodeficiency virus infection.

Methods

Analyze and review the clinical features, diagnosis and treatment of six cases of P. marneffei without human immunodeficiency virus infection at The First Affiliated Hospital of Fujian Medical University.

Results

Two cases were diagnosed in the ENT Department, three cases in the respiratory department and one case in the dermatological department. Penicillium marneffei infection was confirmed by sputum culture, blood culture and tissue biopsy. After definite diagnosis, one refused further treatment, and others showed significant improvement.

Conclusion

Penicilliosis marneffei is insidious onset and easy to be escaped and misdiagnosed. To achieve early diagnosis and appropriate treatment, doubtful cases should be alerted for the diagnoses as P. marneffei.

     

Fine‐scale Spatial Genetic Structure of Ten Dipterocarp Tree Species in a Bornean Rain Forest

Fine‐scale spatial genetic structure is increasingly recognized as an important factor in the studies of tropical forest trees as it influences genetic diversity of local populations. The biologic mechanisms that generate fine‐scale spatial genetic structure are not fully understood. We studied fine‐scale spatial genetic structure in ten coexisting dipterocarp tree species in a Bornean rain forest using microsatellite markers. Six of the ten species showed statistically significant fine‐scale spatial genetic structure. Fine‐scale spatial genetic structure was stronger at smaller spatial scales (≤ 100 m) than at larger spatial scales (> 100 m) for each species. Multiple regression analysis suggested that seed dispersal distance was important at the smaller spatial scale. At the larger scale (> 100 m) and over the entire sample range (0–1000 m), pollinators and spatial distribution of adult trees were more important determinants of fine‐scale spatial genetic structure. Fine‐scale spatial genetic structure was stronger in species pollinated by less mobile small beetles than in species pollinated by the more mobile giant honeybee (Apis dorsata). It was also stronger in species where adult tree distributions were more clumped. The hypothesized mechanisms underlying the negative correlation between clump size and fine‐scale spatial genetic structure were a large overlap among seed shadows and genetic drift within clumped species.     

Anti-<Emphasis Type="Italic">α</Emphasis>-actinin antibodies in relation to new-onset systemic lupus erythematosus and lupus nephritis

The aim of this study is to investigate the role of anti-α-actinin antibodies in patients with new-onset systemic lupus erythematosus (SLE). Thirty-six patients with SLE, 16 of whom had lupus nephritis (LN), and 53 healthy controls were included. The clinical and laboratory parameters of patients were collected from medical records or by questionnaire. Serum anti-α-actinin Abs was measured by competitive enzyme linked immunosorbent assay (ELISA). Our results show that the OD value of serum anti-α-actinin Abs in SLE patients was significantly lower than that in normal controls (1.212 ± 0.244 vs. 1.364 ± 0.202, P = 0.002); seven of 36 SLE patients were seropositive for anti-α-actinin Abs, which was significantly higher than in normal controls (19.4 vs. 3.8%, P = 0.028). There were no significant differences of clinical parameters between the anti-α-actinin Abs-positive patients and the negative patients. The positive rate of the term urine casts, elevated IgM and IgA in anti-α-actinin Abs-positive patients were higher than that in the negative patients. The OD values of serum anti-α-actinin Abs negatively correlated with disease activity (R _s = −0.352, P = 0.035). Anti-α-actinin Abs may be a useful marker of the disease activity of SLE; in addition, it may be used as a complementary parameter to differentiate LN from SLE without nephritis.     

Penicillium marneffei: types and drug susceptibility

The PCR fingerprints of 30 Penicillium marneffei isolates from Chiang Rai in northern Thailand and Bangkok in central Thailand were studied through use of single-nucleotide primers (GACA)₄ and the phage M13 core sequence. Discrimination of fingerprint patterns was based on differences in the number of major bands. The P. marneffei isolates were divided into four types, i.e., A, B, C, and D. Type A was found in two isolates from Chiang Rai (6.7%). Types B and C respectively were found in two (6.7%) and one (3.3%) isolates from Bangkok. The predominate type D (83.3%) was found in isolates obtained from Chiang Rai and Bangkok. The PCR fingerprinting method was found to be useful for the epidemiological study of P. marneffei, a dimorphic opportunistic fungus and an emerging pathogen in the HIV pandemic. In vitro drug susceptibility testing by broth macrodilution to four antifungal agents against the yeast form ofP. marneffei was performed. The MIC ranges for amphotericin B, fluconazole, itraconazole, and ketoconazole were 0.125–0.5, 4.0–8.0, <0.032, and <0.125 μg/ml respectively. This revised version was published online in June 2006 with corrections to the Cover Date.