The decreased number and function of lymphocytes is associated with Penicillium marneffei infection in HIV-negative patients

Background/purposePenicillium marneffei (P. marneffei) infection, which has been traditionally considered as an indicator of immunosuppression, is one of the most common systemic opportunistic infections in patients with AIDS. Recently, more and more P. marneffei infections have been documented in HIV-negative patients without underlying diseases, which challenges the traditional view that P. marneffei infection is an indicator of immunosuppression. We aimed to evaluate the number and function of lymphocytes in HIV-negative patients with P. marneffei infection.Methods15 HIV-negative P. marneffei-infected patients and 18 healthy controls were recruited and investigated. The number and function of lymphocytes were analyzed by flow cytometry.ResultsMost laboratory tests were within the reference ranges, except for a significant increase in total IgE in P. marneffei-infected patients. Lymphocyte subset analysis showed that the number of CD4⁺ T cells and NK cells was significantly decreased in HIV-negative marneffei-infected patients compared with healthy controls. However, almost half of the marneffei-infected patients still had normal levels of lymphocytes. A further analysis of cell function showed that the activation and proliferation of CD4⁺ T cells, the cytotoxicity of CD8⁺ T cells and NK cells, and the cytokine secretion potential of CD4⁺ T cells and NK cells were all impaired, in comparison with healthy controls.ConclusionsP. marneffei infection has to be regarded as an indicator of immunosuppression. A further investigation of cell function is required in patients with opportunistic infection, as the cell function may be impaired in this condition.     

Identification and Purification of Specific Penicillium marneffei Antigens and Their Recognition by Human Immune Sera

Disseminated infection with the dimorphic pathogenic fungus Penicillium marneffei is increasingly seen among patients with AIDS in southeast Asian countries. Previous studies have demonstrated the presence of humoral immune responses to this fungus in patient sera; we have confirmed this work using sera from P. marneffei-infected patients (n = 21) to develop Western blots of P. marneffei cytoplasmic yeast antigen (CYA). P. marneffei CYA was then partially purified by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Immunoenzyme development of the Western blots with pooled sera from patients with P. marneffei infection and with pooled sera from patients with aspergillosis (n = 20), candidiasis (n = 10), cryptococcosis (n = 9), and histoplasmosis (n = 11) revealed three antigens with relative molecular masses of 61, 54, and 50 kDa. These antigens were specifically recognized by the pooled sera from the P. marneffei-infected patients. The 61- and 54-kDa antigens were subsequently purified to homogeneity by preparative gel electrophoresis, and the 50-kDa antigen was partially purified by the same technique. N-terminal amino acid sequencing revealed that the 61-kDa antigen had a strong homology (87% identity) with the antioxidant enzyme catalase. The three antigens were then subjected to SDS-PAGE and Western blotting and to immunoenzyme development with individual patient sera; sera from 86% of P. marneffei-infected patients recognized the 61-kDa antigen, sera from 71% recognized the 54-kDa antigen, and sera from 48% recognized the 50-kDa antigen. These specifically recognized antigens are the first to be purified from P. marneffei and can be used either singly or in combination to detect antibody responses in a large percentage of individuals infected with P. marneffei.     

A novel inhibition ELISA for the detection and monitoring of <Emphasis Type="Italic">Penicillium marneffei</Emphasis> antigen in human serum

The thermally dimorphic fungus Penicillium marneffei is a causative agent of penicilliosis marneffei, a disease considered to be an acquired immune deficiency syndrome (AIDS)-defining illness in Southeast Asia and southern China. We have developed an inhibition enzyme-linked immunosorbent assay (inh-ELISA) incorporating the yeast phase specific mannoprotein-binding monoclonal antibody 4D1 for the detection of P. marneffei infection. In our sample set, the test detected antigenemia in all 45 (100 %) patients with P. marneffei, with a mean antigen concentration of 4.32 μg/ml. No cross-reactivity in this assay was found using serum from 44 additional patients with other fungal infections, such as Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans, as well as 44 patients with bacterial infections, such as Mycobacterium tuberculosis and Streptococcus suis. Additionally, no reactivity occurred using serum from 31 human immunodeficiency virus (HIV)-infected patients without a history of fungal infections and 113 healthy controls residing in endemic areas. To investigate the potential of the inh-ELISA for disease monitoring, we followed the reduction in antigenemia in six patients who clinically responded to itraconazole and P. marneffei was no longer isolated from their blood or tissues. In contrast, we correlated increased concentrations of antigenemia in patients with relapsed P. marneffei infection with the progression of their clinical symptoms and the isolation of P. marneffei from their clinical specimens. In summary, the P. marneffei inh-ELISA is a promising new assay for the rapid diagnosis of P. marneffei, as well as a tool for evaluating clinical response and clearance of the fungus during treatment.     

Effects of Talaromyces marneffei infection on mortality of HIV/AIDS patients in southern China: a retrospective cohort study

Objective

Talaromyces marneffei is an opportunistic infection with high morbidity among human immunodeficiency virus (HIV)/AIDS patients in Southeast Asia and southern China. Its effects on mortality in HIV/AIDS patients has not been clearly elucidated.

Detection of Specific Antibodies to an Antigenic Mannoprotein for Diagnosis of Penicillium marneffei Penicilliosis

The disseminated and progressive fungal disease Penicillium marneffei penicilliosis is one of the most common infectious diseases in AIDS patients in Southeast Asia. To diagnose systemic penicilliosis, we developed an enzyme-linked immunosorbent assay (ELISA)-based antibody test with Mp1p, a purified recombinant antigenic mannoprotein of P. marneffei. Evaluation of the test with guinea pig sera against P. marneffei and other pathogenic fungi indicated that this assay was specific for P. marneffei. Clinical evaluation revealed that high levels of specific antibody were detected in two immunocompetent penicilliosis patients. Furthermore, approximately 80% (14 of 17) of the documented penicilliosis patients with human immunodeficiency virus tested positive for the specific antibody. No false-positive results were found for serum samples from 90 healthy blood donors, 20 patients with typhoid fever, and 55 patients with tuberculosis, indicating a high specificity of the test. Thus, this ELISA-based test for the detection of anti-Mp1p antibody can be of significant value as a diagnostic for penicilliosis.     

Diagnosis of Penicillium marneffei Infection by Quantitation of Urinary Antigen by Using an Enzyme Immunoassay

Penicillium marneffei is a major cause of opportunistic infection in patients with AIDS in north and northeastern Thailand. A method for the quantitation of P. marneffei antigen in urine was developed by using fluorescein isothiocyanate-labelled purified rabbit hyperimmune immunoglobulin G in an enzyme-linked immunosorbent assay. This method was evaluated with 33 patients with culture-proven penicilliosis and 300 controls (52 healthy subjects, 248 hospitalized patients without penicilliosis) from the same area in which penicilliosis is endemic. Urinary antigen was found in all 33 (100%) patients with penicilliosis, with a median titer of 1:20,480. With undiluted samples, 67 (27%) of 248 hospital patients and 3 (6%) of 52 healthy controls were reactive. At a cutoff titer of 1:40, the urine antigen detection assay had a diagnostic sensitivity of 97% and specificity of 98% (positive predictive value, 84%; negative predictive value, 99.7%). This test offers a valuable and rapid method for the diagnosis of penicilliosis in patients with AIDS and could be a useful addition to conventional diagnostic methods in areas in which penicilliosis is endemic.     

Sialic Acid-Dependent Recognition of Laminin by Penicillium marneffei Conidia

Immunofluorescence microscopy demonstrated that laminin bound to the surface of Penicillium marneffei conidia. Attachment of P. marneffei conidia in an adherence assay was inhibited by soluble laminin and anti-laminin antibody. N-Acetylneuraminic acid abolished adherence, indicating an interaction mediated by a sialic acid-specific lectin.     

Different susceptibilities of yeasts and conidia of Penicillium marneffei to nitric oxide (NO)-mediated fungicidal activity of murine macrophages

Penicillium marneffei is an important opportunistic fungal pathogen. Host defence mechanisms against P. marneffei are not fully understood. We investigated the fungicidal activity of murine peritoneal macrophages against two forms of P. marneffei, conidia and yeast cells, and the involvement of the NO-mediated killing system. Peritoneal macrophages suppressed the intracellular growth of P. marneffei yeast cells and conidia. The number of live yeast cells within macrophages was significantly reduced by activation of macrophages by interferon-gamma (IFN-γ), while a similar response was not observed with conidia. IFN-γ-induced macrophage fungicidal activity against yeast cells was mediated by NO and was almost completely inhibited by N^G-monomethyl- L -arginine ( L -NMMA), a competitive inhibitor of NO synthesis, while N^G-monomethyl- D -arginine ( D -NMMA), an optical isomer of L -NMMA, did not show any influence. NO production by macrophages stimulated with IFN-γ was significantly enhanced when these macrophages were cultured with P. marneffei yeast cells, while conidia did not enhance macrophage NO production. Furthermore, yeast cells were more susceptible to the killing effect of chemically generated NO than conidia. Our results indicate that the yeast form of P. marneffei is more sensitive to the fungicidal activity of IFN-γ-stimulated macrophages than conidia, and suggest that the different effects of two forms of P. marneffei on macrophage NO production and their different susceptibilities to NO may be reasons for the present findings.     

First case of subcutaneous infection by Talaromyces marneffei in a renal transplant recipient from India and review of literature

Talaromyces marneffei is one of the endemic mycoses prevalent in South-East Asian region. The infection, which was once considered to be opportunistic infection in HIV-positive patients, is establishing foothold in transplant and immunocompetent population. We report a case of a 41-year-old post-renal transplant female with a travel history to Assam two years back presenting with a subcutaneous lesion on right side of scalp associated with pain and blurring of vision in right eye. Fine-needle aspiration from the scalp lesion showed yeast cells with transverse septation in cytological examination and culture grew Talaromyces marneffei, which was confirmed by sequencing of ITS region. Patient was successfully managed with oral itraconazole 200 mg twice daily for ten months without subsequent recurrence. To our knowledge, this is the first case of subcutaneous infection by T. marneffei in a renal transplant recipient from India.     

An update on cutaneous infections

The spectrum of infectious diseases with potential skin involvement, either primary or secondary to systemic disease, is vast. Unusual host inflammatory responses may be elicited by certain pathogens, including reactions with an inherent capacity for misdiagnosis as neoplasia. Current, emerging, re-emerging and/or recently recognized pathogens or infections of major significance include carbapenem-resistant gram-negative bacterial infections, Ebola virus disease, measles, Mycobacterium lepromatosis as a second aetiological agent of leprosy, and Emergomyces africanus infection among those living with HIV/AIDS. The era of molecular microbiology has also seen recent changes in taxonomy, including Talaromyces marneffei (formerly Penicillium marneffei) and Cutibacterium acnes (formerly Propionobacterium acnes). Precise speciation by PCR on formalin-fixed, paraffin embedded tissue, however, remains a challenge despite recent advances in molecular microbiology. Finally, one must remain cognizant of the wide range of potential infective manifestations of the immune reconstitution inflammatory syndrome in HIV-infected individuals receiving antiretroviral therapy.     

Immune Restoration Syndrome with disseminated Penicillium marneffei and Cytomegalovirus co-infections in an AIDS patient

Background  

Penicillium marneffei is a dimorphic fungus, endemic in South-east Asia. The fungus causes severe disease in immunocompromised patients such as AIDS. However, no case of immune restoration disease of Penicillium marneffei is reported in literature from a non-endemic area.     

Detection of Mycoplasma fermentans DNA from lymph nodes of acquired immunodeficiency syndrome patients

Biopsy samples from seven patients with acquired immunodeficiency syndrome were screened for Mycoplasma fermentans, M. pneumoniae and M. genitalium infection by the polymerase chain reaction. M. fermentans DNA was detected in four patients. Various tissues were evaluated and the mycoplasma were mainly detected from lymph nodes. Moreover, mycoplasma genus-specific DNA was isolated from peripheral blood mononuclear cells of asymptomatic human immunodeficiency virus(HIV)-infected individuals (two of 31 HIV-infected individuals). These data suggest that mycoplasma infection in AIDS patients is not uncommon.     

B Cell Production of Tumor Necrosis Factor in Response to Pneumocystis murina Infection in Mice

Pneumocystis species are opportunistic fungal pathogens that induce tumor necrosis factor (TNF) production by alveolar macrophages. Here we report that B cells from the draining lymph nodes as well as lung CD4⁺ T cells are important producers of TNF upon Pneumocystis murina infection. To determine the importance of B cell-derived TNF in the primary response to P. murina, we generated bone marrow chimeras whose B cells were unable to produce TNF. The lung P. murina burden at 10 days postinfection in TNF knockout (TNFKO) chimeras was significantly higher than that in wild-type (WT) chimeras, which corresponded to reduced numbers of activated CD4⁺ T cells in the lungs at this early time point. Furthermore, CD4⁺ T cells isolated from P. murina-infected TNFKO chimeras were unable to stimulate clearance of P. murina upon adoptive transfer to recombinase-deficient (RAG1KO) hosts. Together, these data indicate that B cell-derived TNF plays an important function in promoting CD4⁺ T cell expansion and production of TNF and facilitating protection against P. murina infection.     

Detection of Pneumocystis jirovecii by quantitative real-time PCR in oral rinses from Pneumocystis pneumonia asymptomatic human immunodeficiency virus patients

Pneumocystis pneumonia (PCP) is a potentially life-threatening fungal infection usually seen in immunocompromised patients. Pneumocystis jirovecii can be easily detected from oral rinse samples in HIV patients with suspected PCP. In this study, a quantitative real-time PCR assay was used to establish the frequency of detection of P. jirovecii in oral rinses from HIV patients without respiratory symptoms or suspicion of PCP. Two saline oral rinses were collected from 100 ambulant HIV patients and from 60 COPD patients (comparator group). Four HIV patients were positive for P. jirovecii. In three patients, the first sample was positive and in one the second one was positive. One of these patients was on PCP prophylaxis and had a CD4⁺ count of 76 cells/mm³. The mean CD4⁺ count for all patients was 527 cells/mm³. All qRT-PCR test results for the COPD patients were negative. No patient developed PCP at six months follow-up. The qRT-PCR assay can be used to detect P. jirovecii DNA in oral rinse samples from HIV patients without evident clinical symptoms, however the oral carriage of this fungus was rare in our cohort of patients. In conclusion, although rare, a positive oral rinse P. jirovecii result may reflect colonisation, in particular in patients with HIV. This needs to be kept in mind when using oral rinses and qRT-PCR in the diagnosis of P. jirovecii infection.     

Disseminated penicilliosis marneffei in immunocompetent patients: A report of two cases

Disseminated penicilliosis marneffei is rarely seen in immunocompetent persons. We report here two cases of disseminated penicilliosis marneffei in immunocompetent hosts. Penicillium marneffei disseminated to the brain in one patient and to the bone marrow in the other patient. Both patients received amphotericin B liposome. The cases illustrate the importance of considering penicilliosis marneffei as causes of systemic infections in immunocompetent patients.     

Dimorphic fungal osteoarticular infections

The objective of this investigation was to review the clinical manifestations, management, and outcome of osteoarticular infections caused by dimorphic fungi. We exhaustively reviewed reports of bone and joint infections caused by dimorphic fungi published between 1970 and 2012. Underlying conditions, microbiological features, histological characteristics, clinical manifestations, antifungal therapy, and outcome were analyzed in 222 evaluable cases. Among 222 proven cases (median age 41 years [interquartile range (IQR) 26–57]), 73 % had no predisposing condition. Histopathology performed in 128 (57 %) cases and culture in 170 confirmed diagnosis in 63 % and 98 % of the cases, respectively. Diagnosis was obtained from an extra-osteoarticular site in 16 cases. The median diagnostic time was 175 days (IQR 60–365). Sporothrix schenckii was the most frequent pathogen (n?=?84), followed by Coccidioides immitis (n?=?47), Blastomyces dermatitidis (n?=?44), Histoplasma capsulatum (n?=?18), Paracoccidioides brasiliensis (n?=?16), and Penicillium marneffei (n?=?13). Arthritis occurred in 87 (58 %) cases and osteomyelitis in 64 (42 %), including 19 vertebral osteomyelitis. Dissemination was reported in 123 (55 %) cases. Systemic antifungal agents were used in 216 (97 %) patients and in combination with surgery in 129 (60 %). Following the Infectious Diseases Society of America (IDSA) guidelines, a successful initial medical strategy was observed in 97/116 (84 %) evaluable cases. The overall mortality was 6 %, and was highest for P. marneffei (38.5 %). This study demonstrates that dimorphic osteoarticular infections have distinctive clinical presentations, occur predominantly in apparently immunocompetent patients, develop often during disseminated disease, and may require surgical intervention.     

Candida tropicalis and Penicillium marneffei Mixed Fungaemia in a Patient with Waldenström's Macroglobulinaemia

A patient with Waldenström's macroglobulinaemia who had previously been treated with cladribine presented with septic arthritis caused by Escherichia coli. The patient's condition subsequently deteriorated, and he succumbed to pneumonia and mixed fungaemia due to Candida tropicalis and Penicillium marneffei. Profound lymphopenia coexisted with fungaemia. This is the first reported case of mixed Penicillium marneffei and Candida fungaemia and penicilliosis marneffei in a patient with Waldenström's macroglobulinaemia. Penicilliosis marneffei should be considered as a potential complication in patients with markedly impaired cell-mediated immunity who have travelled to or resided in endemic areas. Patients who have undergone therapy with purine nucleoside analogues such as fludarabine and cladribine are also at risk.     

MP1 Encodes an Abundant and Highly Antigenic Cell Wall Mannoprotein in the Pathogenic Fungus Penicillium marneffei

We cloned the MP1 gene, which encodes an abundant antigenic cell wall mannoprotein from the dimorphic pathogenic fungus Penicillium marneffei. MP1 is a unique gene without homologs in sequence databases. It codes for a protein, Mp1p, of 462 amino acid residues, with a few sequence features that are present in several cell wall proteins of Saccharomyces cerevisiae and Candida albicans. It contains two putative N glycosylation sites, a serine- and threonine-rich region for O glycosylation, a signal peptide, and a putative glycosylphosphatidylinositol attachment signal sequence. Specific anti-Mp1p antibody was generated with recombinant Mp1p protein purified from Escherichia coli to allow further characterization of Mp1p. Western blot analysis with anti-Mp1p antibody revealed that Mp1p has predominant bands with molecular masses of 58 and 90 kDa and that it belongs to a group of cell wall proteins that can be readily removed from yeast cell surfaces by glucanase digestion. In addition, Mp1p is an abundant yeast glycoprotein and has high affinity for concanavalin A, a characteristic indicative of a mannoprotein. Furthermore, ultrastructural analysis with immunogold staining indicated that Mp1p is present in the cell walls of the yeast, hyphae, and conidia of P. marneffei. Finally, it was observed that infected patients develop a specific antibody response against Mp1p, suggesting that this protein represents a good cell surface target for host humoral immunity.     

The cellular transfer of immunity to Nippostrongylus brasiliensis in inbred rats (Lewis strain)

Mesenteric lymph node cells obtained from highly inbred donor rats (Lewis strain), resistant to Nippostrongylus brasiliensis infection, were syngeneically transferred by intravenous injection into previously uninfected recipients. The adoptively immunized recipients were then challenged with either 1500 or 3000 third stage N. brasiliensis larvae on the day of cell transfer. The degree of resistance transferred was assessed by monitoring daily faecal egg output, differential worm burdens on days 6 and 10 of infection and the number of eggs per uterus in gravid worms.

The syngeneic transfer of 100 × 10⁶ immune mesenteric lymph node cells invariably resulted in suppression of egg production, a two- to four-fold reduction in the number of eggs per uterus in gravid females and rejection of at least 75 per cent of adult worms by days 6 and 10 of infection.

It was also noted that mesenteric lymph node cells obtained from donors on day 15 of a primary infection were more effective than those obtained from donors immunized by multiple infections.

Immune cells transferred from donors on day 4 of infection were equally effective with those transferred on day 0. However, immune cells transferred on or after day 10 of infection had little or no effect and this shows that the parasite is less susceptible to an attack mounted by the transferred cells during the later stages of infection.

     

Antibody-free rapid diagnosis of malaria in whole blood with surface-enhanced Raman Spectroscopy using Nanostructured Gold Substrate

PurposeThe aim of this study is to establish a rapid antibody-free diagnostic method of malaria infection with Plasmodium falciparum and Plasmodium vivax in whole blood with Surface-enhanced Raman Spectroscopy using Nanostructured Gold Substrate.Materials and methodsThe blood samples collected from patients were first lysed and centrifuged before dropping on the gold nano-structure (AuNS) substrate. Malaria diagnosis was performed by detecting Raman peaks from Surface Enhanced Raman Spectroscopy (SERS) with a 532 nm laser excitation.ResultsRaman peaks at 1370 cm⁻¹, 1570 cm⁻¹, and 1627 cm⁻¹, known to have high specificity against interference from other mosquito-borne diseases such as Dengue and West Nile virus infection, were selected as the fingerprint markers associated with P. falciparum and P. vivax infection. The limit of detection was 10⁻⁵ dilution, corresponding to the concentration of parasitized blood cells of 100/mL. A total number of 25 clinical samples, including 5 from patients with P. falciparum infection, 10 with P. vivax infection and 10 from healthy volunteers, were evaluated to support its clinical practical use. The whole assay on malaria detection took 30 min to complete.ConclusionsWhile the samples analyzed in this work have strong clinical relevance, we have clearly demonstrated that sensitive malaria detection using AuNS-SERS is a practical direction for rapid in-field diagnosis of malaria infection.