侵袭性真菌病的诊断：现状与展望

近二十年来,医学科学很多领域都取得重大进步。但全球范围内,侵袭性真菌病的发病率及死亡率却仍明显上升,严重威胁人类健康。侵袭性真菌病发病隐匿、临床表现不典型、治疗手段有限、病死率与致残率高,早期、特异的诊断对于改善预后意义重大。目前,以培养、病理为代表的形态学诊断方法虽有局限,但仍是侵袭性真菌病诊断的金标准;以G试验、GM试验、高分辨率CT为代表的新兴血清学及影像学诊断方法值得在临床大力推广;而以PCR技术为基础的核酸诊断技术方法前景光明,但其临床应用之路却仍任重而道远。联合使用并不断改良现有培养、病理等形态学诊断方法、血清学方法及先进影像学技术是提高侵袭性真菌病诊断水平的现实最佳途径。     

Determination of PCR efficiency in chelex-100 purified clinical samples and comparison of real-time quantitative PCR and conventional PCR for detection of Chlamydia pneumoniae

Background  

Chlamydia pneumoniae infection has been detected by serological methods, but PCR is gaining more interest. A number of different PCR assays have been developed and some are used in combination with serology for diagnosis. Real-time PCR could be an attractive new PCR method; therefore it must be evaluated and compared to conventional PCR methods.     

The Future of Fungal Serology

The incidence of invasive fungal infections continues to grow. Early and rapid diagnosis is essential to prevent morbidity and mortality. The number of assays available for the detection of fungal antigens in human body fluids are increasing in number and becoming part of the basic diagnostic workup for many fungal infections. Detection of specific antibody has been an important component in the diagnosis of fungal infections. Complement fixation and immunodiffusion continue to be the gold standard for antibody detection but are complex to perform, require extensive expertise, and are mostly performed in reference labs. Newer assays are being developed to reduce turn-around time, but have not been fully evaluated. A challenge for improving serologic assays is to move from crude antigens and polyclonal antibodies to purified and/or recombinant antigens and monoclonal antibodies, while retaining good sensitivity and specificity. Recent developments using lateral flow methodology have provided novel point-of-care antigen assays requiring little technical expertise. Such innovative techniques will help to keep the future of fungal serology bright.     

An Approach to Suspected Invasive Fungal Infection in Patients with Hematologic Malignancy and HCT Recipients with Persistent Neutropenic Fever Despite Mold-Active Prophylaxis

Purpose of Review

This review offers an approach to managing suspected invasive fungal infection (IFI) in a febrile neutropenic patient with hematologic malignancy or hematopoietic cell transplantation (HCT) while on mold-active prophylaxis. We take into consideration host characteristics, new diagnostic tools, and available therapeutics.

Recent Findings

Despite use of anti-Aspergillus prophylactic agents, invasive aspergillosis is the most commonly reported IFI breaking through common prophylactic agents including the newest azole, isavuconazole. While more fungal diagnostic modalities are available, how to best incorporate them in the work-up of IFI remains unclear, while sensitivity of any particular fungal biomarker or molecular test is low.

Summary

In a febrile neutropenic patient with hematologic malignancy or HCT and suspected IFI, consider particularly invasive aspergillosis, regardless of the mold-active prophylactic agent. Early diagnosis and intervention are especially important to a favorable outcome; treatment is directed based on the suspected IFI syndrome and suspected organism. Switching azoles, consideration of combination therapy, and reducing immunosuppression are proposed strategies for the management of breakthrough IFI, while surgical debridement remains crucial for Mucormycoses. More study is needed into the optimal antifungal approach in these clinical scenarios. Meanwhile, therapeutic drug monitoring and attention to drug-drug interactions are encouraged.

     

A MIQE-compliant real-time PCR assay for Aspergillus detection

The polymerase chain reaction (PCR) is widely used as a diagnostic tool in clinical laboratories and is particularly effective for detecting and identifying infectious agents for which routine culture and microscopy methods are inadequate. Invasive fungal disease (IFD) is a major cause of morbidity and mortality in immunosuppressed patients, and optimal diagnostic criteria are contentious. Although PCR-based methods have long been used for the diagnosis of invasive aspergillosis (IA), variable performance in clinical practice has limited their value. This shortcoming is a consequence of differing sample selection, collection and preparation protocols coupled with a lack of standardisation of the PCR itself. Furthermore, it has become clear that the performance of PCR-based assays in general is compromised by the inadequacy of experimental controls, insufficient optimisation of assay performance as well as lack of transparency in reporting experimental details. The recently published "Minimum Information for the publication of real-time Quantitative PCR Experiments" (MIQE) guidelines provide a blueprint for good PCR assay design and unambiguous reporting of experimental detail and results. We report the first real-time quantitative PCR (qPCR) assay targeting Aspergillus species that has been designed, optimised and validated in strict compliance with the MIQE guidelines. The hydrolysis probe-based assay, designed to target the 18S rRNA DNA sequence of Aspergillus species, has an efficiency of 100% (range 95-107%), a dynamic range of at least six orders of magnitude and limits of quantification and detection of 6 and 0.6 Aspergillus fumigatus genomes, respectively. It does not amplify Candida, Scedosporium, Fusarium or Rhizopus species and its clinical sensitivity is demonstrated in histological material from proven IA cases, as well as concordant PCR and galactomannan data in matched broncho-alveolar lavage and blood samples. The robustness, specificity and sensitivity of this assay make it an ideal molecular diagnostic tool for clinical use.     

Clinical and diagnostic management of toxoplasmosis in the immunocompromised patient

With the advent of the highly active antiretroviral therapy (HAART), the natural course of HIV infection has markedly changed and opportunistic infections including toxoplasmosis have declined and modified in presentation, outcome and incidence. However, TE is a major cause of morbidity and mortality especially in resource-poor settings but also a common neurological complication in some countries despite the availability of HAART and effective prophylaxis. In most cases toxoplasmosis occurs in brain and toxoplasmic encephalitis (TE) is the most common presentation of toxoplasmosis in immunocompromised patients with or without AIDS. The need of a definitive diagnosis is substantial because other brain diseases could share similar findings. Rapid and specific diagnosis is thus crucial as early treatment may improve the clinical outcome. Classical serological diagnosis is often inconclusive as immunodeficient individuals fail to produce significant titres of specific antibodies. Polymerase chain reaction (PCR) has a high diagnostic value in the acute disease, but like many 'in-house' PCR assays, suffers from lack of standardization and variable performance according to the laboratory. Molecular diagnosis of toxoplasmosis can be improved by performing real-time PCR protocols. This article summarises the clinical manifestations, diagnostic procedures and management strategies for this condition.     

Challenges in the Diagnosis of Invasive Fungal Infections in Immunocompromised Hosts

Purpose of review

The expanding population of immunocompromised patients coupled with the recognition of a growing number of different species of fungi responsible for diseases in such hosts makes the diagnosis of invasive fungal infection (IFI) a challenging task. The recent advances and challenges in the diagnosis of IFI in the setting of immunocompromised hosts are reviewed. The advantages and limitations of histopathology and the role of culture-independent methods, such as those based on the use of nucleic acids applied to fresh and formalin-fixed, paraffin-embedded sections, besides culture- and non-culture-based diagnostic methods, to obtain a timely and correct diagnosis of IFI are highlighted.

Recent findings

The therapeutic implications of identifying the genus and species of the fungus present in the specimen with the molecular diagnostics applied to tissue specimens are reviewed. No method alone is efficient in correctly identifying fungi and it is essential to combine the traditional histochemical staining with molecular methods to achieve a rapid and genus-/species-specific diagnosis of IFI.

Summary

We review the recent findings and challenges in the hystopathologic diagnosis of IFI in the setting of immunocompromised hosts. Non method alone is efficient in correctly identify fungi and pathologists should combine classic staining with molecular methods to achieve a rapid and genus/species fungal diagnosis.

     

Epidemiology of invasive fungal infection in solid organ transplant

Despite advances made in the last decades, invasive fungal infections (IFI) continue to be a major cause of morbidity and mortality in solid organ transplant recipients. The most common pathogens causing IFI are Candida species, followed by Aspergillus and Cryptococcus. A shift in the epidemiology of IFI has been reported in the last few years. Non-Candida albicans Candida species and non-Aspergillus filamentous moulds have been increasingly observed in transplant patients. A change in the IFI onset time has also been described recently. In the RESITRA (Spanish Network of Infection in Transplantation) study, at least 50% of invasive aspergillosis (IA) infections and 40% of invasive Candida infections had been observed after 180 days of transplant. Some cases of cryptococcal infection, traditionally considered as a late onset infection, have been observed in the early post transplant period. Mortality due to IFI is still high, particularly in patients with IA. However, the progressive improvement achieved in diagnosis and prevention of IFI has led to a lower mortality rate.     

Overview of diagnostic and detection methods for legionellosis and Legionella spp.

Since 1977, the diagnostic tools for Legionnaires' disease have been based on culture and serological investigations. Both methods require considerable time to produce results and have 'low' to 'reasonable' sensitivity. Since the introduction of urinary antigen tests in the mid 1990s, underdiagnosis has diminished and mortality has declined, thanks to early diagnosis. To obtain the most accurate diagnosis, culture, serological investigation, and urinary antigen testing should all be performed. In the last decade, advances in polymerase chain reaction technology allowed the development of assays detecting Legionella nucleic acids in clinical and environmental samples. Thus far, only widely varying results with relatively small series have been reported. Furthermore, these assays which are still labour intensive and complicated are not yet practicable for the average medical and/or environmental microbiological laboratory.     

Diagnostic Values and Limitations of (1,3)-β-<Emphasis Type="SmallCaps">d</Emphasis>-Glucans and Galactomannan Assays for Invasive Fungal Infection in Patients Admitted to Pediatric Intensive Care Unit

The relationship among (1,3)-β-d-glucans (BG), galactomannan (GM), and the risk of developing invasive fungal infections (IFI) has been observed in adult ICU and in children with hematological malignancies. Only scant data evaluated the value of BG/GM assays for diagnosis of IFI in patients with nonhematological diseases in pediatric intensive care unit (PICU). In this study, we assessed the diagnostic value of these markers for IFI in PICU. The records of 230 patients were retrospectively evaluated. Out of 117 patients (7 proven, 23 probable, and 87 cases without evidence of IFI) performed GM and BG assays. The results showed many factors were associated with false-positive test results. Patients who aged over 3 years had higher levels of GM and BG than younger infants. The levels of BG were higher in subjects with dairy, human blood products, antibiotics, and corticosteroids therapy than in cases without these treatments. Unlike BG assay, GM assay was less susceptible to above-mentioned factors expect blood products. The levels of BG and GM in IFI cases were dramatically higher than in controls. The diagnostic performance of these assays showed that GM assay had better results when compared with BG assay. On the whole, negative predictive value in both GM and BG assays was dramatically higher than other diagnostic parameters. In conclusion, BG assay was highly susceptible to many factors, and GM assay could be useful for diagnosis of IFI for its high sensitivity, but the over benefit of this assay limited in its inadequate specificity. The comparative advantage of BG and BG assays lied in excluding IFI in non-hematological PICU patients.     

La infección fúngica en el paciente pediátrico inmunodeprimido

In recent years, immunodeficiency condition has experienced a rise among children, who are at risk of invasive fungal infections (IFI) due to their health condition. Cancer, non-malignant hematological diseases, as primary immunodeficiencies, hematopoietic stem cell transplantation (HSCT), extreme prematurity, or critically ill condition in Pediatric Intensive Care Unit (PICU) are some immunosuppressive situations in children. The use of oncologic therapies, including immunotherapy and monoclonal antibodies, for the treatment of the aforementioned health conditions has led to an increase in morbidity and mortality rates of IFI in children.The underlying diseases and their management, comorbidities, the diagnostic tests used (both molecular and imaging), as well as the treatment used can be significantly different between adult patients and children admitted to PICU or with cancer. In pediatrics, the treatment of IFI is based primarily on pharmacokinetic studies performed in adults. In higher risk patients prophylaxis should be considered and, in the case of an IFI diagnosis, an antifungal treatment should be administered as early as possible, supported by the reversion of the immune dysfunction and surgery when appropriate.     

Update on the contribution of galactomannan for the diagnosis of invasive aspergillosis

The diagnosis of invasive fungal infections (IFI) remains a challenge, particularly for diseases caused by filamentous fungi such as Aspergillus species. Unfortunately, many patients affected by these conditions are not identified before autopsy. Therefore, there is a need for new diagnostic methods for IFI. Galactomannan is a soluble antigen released during hyphal growth in tissues. A commercially available sandwich ELISA assay that detects galactomannan has been used in Europe for many years and is now approved for use in the USA. The test has an excellent negative predictive value in the detection of invasive aspergillosis (IA) in high-risk patients. In addition, it is more sensitive than culture and allows IA to be diagnosed before clinical manifestations occur. However, false-negative and false-positive results in certain populations are the main limitations to its use. The purpose of this review is to summarize the current knowledge about galactomannan testing in patients at risk for IA.     

Fungal molecular diagnostics: a mini review

Conventional methods to identify fungi have often relied on identification of disease symptoms, isolation and culturing of environmental organisms, and laboratory identification by morphology and biochemical tests. Although these methods are still fundamental there is an increasing move towards molecular diagnostics of fungi in all fields. In this review, some of the molecular approaches to fungal diagnostics based on polymerase chain reaction (PCR) and DNA/RNA probe technology are discussed. This includes several technological advances in PCR-based methods for the detection, identification and quantification of fungi including real-time PCR which has been successfully used to provide rapid, quantitative data on fungal species from environmental samples. PCR and probe based methods have provided new tools for the enumeration of fungal species, but it is still necessary to combine the new technology with more conventional methods to gain a fuller understanding of interactions occurring in the environment. Since its introduction in the mid 1980's PCR has provided many molecular diagnostic tools, some of which are discussed within this review, and with the advances in micro-array technology and real-time PCR methods the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual fungal species but also on whole communities.     

Diagnóstico microbiológico de las micosis invasoras

The prognosis of invasive fungal infections (IFI) depends on the speed of diagnosis and treatment. Conventional diagnostic methods are of low sensitivity, laborious and too slow, leading to the need for new, faster, and more efficient diagnostic strategies.There are several techniques for diagnosing a candidemia that are faster than the conventional blood culture (BC). Once yeast growth in BC is detected, species identification can be speeded up by mass spectrometry (30 minutes), commercialised molecular techniques (60-80 minutes) or fluorescent in situ hybridization (90 minutes). The combined detection of biomarkers (antimicellium, mannan and anti-mannan or β-glucan) has shown to be of greater use than their individual use. Commercialised nucleic acid amplification techniques (Septifast®, T2Candida®) are very reliable alternatives to BC. The detection of the capsular antigen of Cryptococcus, by means of latex agglutination or immuno-chromatography, is a valuable technique for cryptococcosis diagnosis.Direct microscopic examination and culture of representative specimens is used for the conventional diagnosis of IFI by filamentous fungi. Detection of galactomannan and β-glucan are considered diagnostic criteria for probable invasive aspergillosis and probable IFI, respectively, despite the lack of specificity of the latter. The detection of fungal volatile organic compounds in breath is an interesting diagnostic strategy in pulmonary infections. Although widely used, nucleic acid detection techniques are not considered diagnostic criteria for IFIs caused by moulds in consensus documents, due to their lack of standardisation. However, they are the only alternative to culture methods in invasive infections by Scedosporium/Lomentospora, Fusarium, zygomycetes, or dematiaceous fungi.     

Epidemiologic Research of Invasive Fungal Infections Using Large Healthcare Databases

Current Fungal Infection Reports - Invasive fungal infections (IFI) have become an important cause of morbidity and mortality over the past several decades, especially among immunocompromised...     

Review of Epidemiology, Diagnosis, and Treatment of Invasive Mould Infections in Allogeneic Hematopoietic Stem Cell Transplant Recipients

Invasive mould infections are a major cause of morbidity and mortality in hematopoietic stem cell transplant recipients (HSCT). Allogeneic HSCT recipients are at substantially higher risk than autologous HSCT recipients. Although neutropenia following the conditioning regimen remains an important risk factor for opportunistic fungal infections, most cases of invasive mould infection in allogeneic HSCT recipients occur after neutrophil recovery in the setting of potent immunosuppressive therapy for graft-versus-host disease. Invasive aspergillosis is the most common mould infection. However, there has been an increased incidence of less common non-Aspergillus moulds that include zygomycetes, Fusarium sp., and Scedosporium sp. Reflecting a key need, important advances have been made in the antifungal armamentarium. Voriconazole has become a new standard of care as primary therapy for invasive aspergillosis based on superiority over amphotericin B. There is significant interest in combination therapy for invasive aspergillosis pairing voriconazole or an amphotericin B formulation with an echinocandin. There have also been advances in novel diagnostic methods that facilitate early detection of invasive fungal infections that include galactomannan and beta-glucan antigen detection and PCR using fungal specific primers. We review the epidemiology, diagnosis, and management of invasive mould infection in HSCT, with a focus on allogeneic recipients. We also discuss options for prevention and early treatment of invasive mould infections.     

Candidemia persistente en una paciente receptora de trasplante renopancreático

BackgroundIn recent decades, there has been an increase in the survival of recipients of solid organ trans-plants related to the improvement of the surgical technique, the introduction of protocols for immunosu-ppressive therapy, and the use of antimicrobial prophylaxis. Nonetheless, invasive fungal infection (IFI) is currently the major cause of morbidity and mortality in this group of patients. Invasive candidiasis is the most common IFI found after renal transplantation and is usually associated with total parenteral nutrition, broad-spectrum antibiotic therapy and abdominal surgery.Clinical caseWe report the case of a recent kidney-pancreas transplant recipient who developed a persis-tent catheter-related candidemia caused by Candida glabrata. The patient was treated with anidulafungin and had a good clinical course with no significant drug interactions.We discuss the possible causes and diagnostic and therapeutic alternatives of this kind of infection.     

Evaluation of a polymerase chain reaction assay for the diagnosis of bovine trypanosomiasis and epidemiological surveillance in Bolivia

Background  

Sporadic outbreaks of bovine trypanosomiasis have been reported in Bolivia since 1996 when T. vivax and T. evansi were identified for the first time by parasitological means. However, comprehensive epidemiological information concerning T. vivax and T. evansi in the country is lacking. Current parasitological and serological diagnostic methods for trypanosomiasis have important limitations either in their sensitivity or specificity, which can result in unreliable data when applied in epidemiological studies. PCR assays are a recently developed procedure that might help to overcome the constraints of parasitological and serological assays. Therefore, the objective of this study was to evaluate PCR assays as a diagnostic tool for epidemiological studies in Bolivia.     

Evolución de la infección fúngica invasora en los últimos 30 años

Clinical mycology is in continuous development. The appearance of new clinical guidelines has made it possible to improve the approach to opportunistic fungal infections, especially in immunosuppressed patients (oncohematological and/or transplant recipients). At the same time, the development of new diagnostic tools and new antifungals with a greater spectrum of action and fewer side effects have led to faster diagnoses and treatments that are more effective. Along with these advances, there has been a change in the epidemiology of invasive fungal infection (IFI), with the appearance of new patients (e.g., COPD, liver cirrhosis, post-influenza) and new microorganisms (Candida auris, Lomentospora prolificans, mucorales), and resistant fungi (isolates of Aspergillus resistant to azoles) which the clinician must take into account when choosing the treatment of a patient with an IFI. In this paper we will briefly review the advances in recent decades and the emerging problems.     

Diagnosis and seroepidemiology of Neospora caninum-associated bovine abortion

A round table was conducted at the VIIIth International Coccidiosis Conference on Neospora diagnosis with particular emphasis on strategies to diagnose bovine abortion. The strength and weakness of different assays for Neospora caninum infection and whether these methods have resulted in the overdiagnosis of neosporosis was discussed. It was evident that each diagnostic method, namely histology, immunohistochemistry, molecular detection and serological assays were, under certain circumstances, valuable in assessing the role N. caninum in abortion. Histological, immunohistochemical and molecular detection assays are of outstanding importance for the examination of tissues of aborted foetuses. While histology and immunohistochemistry allow direct assessment of pathomorphological changes caused by infection, molecular detection assays such as PCR are superior because of higher sensitivity and specificity in identifying N. caninum in foetal tissues. Serological tests, such as ELISA, are useful in determining whether an animal has been infected with N. caninum. Seroepidemiological approaches allow one to assess an abortion problem at a herd level and when used in conjunction with certain statistical methods are able to confirm a suspected N. caninum-associated abortion.