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.     

Impact of HIV Infection Status on Interpretation of Quantitative PCR for Detection of Pneumocystis jirovecii

Quantitative PCR (qPCR) is now a key diagnostic tool for Pneumocystis pneumonia. However, cutoffs to distinguish between infected and colonized patients according to their HIV status have not yet been determined. According to clinical, radiological, and biological data, we retrospectively classified bronchoalveolar lavage (BAL) samples subjected to qPCR over a 3-year period into four categories, i.e., definite PCP, probable PCP, Pneumocystis colonization, and no infection. Fungal burden was then analyzed according to the HIV status of the patients. Among 1,212 episodes of pneumonia screened in immunocompromised patients, 52 and 27 HIV-positive patients were diagnosed with a definite and probable PCP, whereas 4 and 22 HIV-negative patients had definite and probable PCP, respectively. Among patients with definite or a probable PCP, HIV-negative patients had a significantly lower burden than HIV-positive patients (P < 10⁻⁴). In both groups, the median fungal burden was significantly higher in patients with definite PCP than in colonized patients. A single cutoff at 1.5 × 10⁴ copies/ml allowed to differentiate colonized and infected HIV-positive patients with 100% sensitivity and specificity. In HIV-negative patients, cutoff values of 2.87 × 10⁴ and 3.39 × 10³ copies/ml resulted in 100% specificity and sensitivity, respectively. Using cutoffs determined for the whole population would have led us to set aside the diagnosis of PCP in 9 HIV-negative patients with definite or probable PCP. qPCR appeared to be the most sensitive test to detect Pneumocystis in BAL samples. However, because of lower inocula in HIV-negative patients, different cutoffs must be used according to the HIV status to differentiate between colonized and infected patients.     

Recent Advances in Our Understanding of the Environmental,Epidemiological, Immunological,and Clinical Dimensions of Coccidioidomycosis

SUMMARY

Coccidioidomycosis is the endemic mycosis caused by the fungal pathogens Coccidioides immitis and C. posadasii. This review is a summary of the recent advances that have been made in the understanding of this pathogen, including its mycology, genetics, and niche in the environment. Updates on the epidemiology of the organism emphasize that it is a continuing, significant problem in areas of endemicity. For a variety of reasons, the number of reported coccidioidal infections has increased dramatically over the past decade. While continual improvements in the fields of organ transplantation and management of autoimmune disorders and patients with HIV have led to dilemmas with concurrent infection with coccidioidomycosis, they have also led to advances in the understanding of the human immune response to infection. There have been some advances in therapeutics with the increased use of newer azoles. Lastly, there is an overview of the ongoing search for a preventative vaccine.     

Mycology of Onychomycosis

Onychomycosis is a fungal infection that can occur within the nails of the fingers and the toes. These infections can lead to discoloration and thickening of the nails, as well as separation of the nails from the bed and their splitting, and ultimately nail destruction. Although predominantly caused by dermatophytes, which have keratinolytic properties, several other groups of fungi can also cause this type of infection, including nondermatophyte molds (both hyaline and dematiaceous) and yeasts, such as Candida species. Proper identification of the etiologic agent is important, as it may influence the treatment of these infections. Here, we review the mycology of onychomycosis and describe recent changes in fungal taxonomy that have occurred with several of these fungi.     

Keeping up with emerging fungal infections

Emerging fungal infections are an important component of infectious disease diagnosis and public health assessment. These infections cause significant morbidity and mortality and can be difficult to diagnose. Recent episodes of fungal infections are presented, including the ongoing Cryptococcus gattii outbreak in British Columbia, Canada, and the northwestern United States; the Apophysomyces trapeziformis infections associated with the Joplin, Missouri, tornado in 2011; the emerging unusual Aspergillus infections associated with antifungal prophylaxis; and the ongoing fungal meningitis outbreak associated with contaminated steroids. Lessons learned from these episodes are discussed in order to provide a platform for illustrating dilemmas in mycology. Useful tools to help the microbiology laboratory stay informed about emerging fungal infections are also discussed.     

Emerging and reemerging fungal infections

Fungal infections throughout the world appear to be increasing. This may in part be due to the increase in the population of patients that are susceptible to otherwise rare fungal infections resulting from the use of immune modulating procedures such as hematopoietic stem cell transplants and drugs like tissue necrosis factor antagonists. Histoplasma capsulatum, an endemic fungus throughout North and South America, is reemerging among HIV+ patients in Central and South America and among patients taking tissue necrosis factor antagonists and other biologics in North America. Fusarium species, a relatively rare fungal infection, is reemerging worldwide in the immunocompromised populations, especially those who are neutropenic like hematopoietic stem cell transplant recipients. A new yeast species is currently emerging worldwide: Candida auris, unknown just a decade ago. It is causing large healthcare-associated outbreaks on four continents and is spreading throughout the world through patient travel. In this review the epidemiology, pathology, detection and treatment of these three emerging and reemerging fungi will be discussed.     

Anti-Candida antibodies and candidemia in ninety patients with HIV/AIDS and cancer

Immunocompromised states such as HIV/AIDS and cancer render the host susceptible to a wide spectrum of infections including fungal infections. The role of humoral immune response in immunity against fungal pathogens such as Candida in immunocompromised individuals such as HIV patients has been questionable owing to the low level or absence of antibodies in these patients. The objective of this study was to analyse the seroprevalence of anti-Candida antibodies in conjunction with occurrence of candidemia in immunocompromised (HIV and cancer) patients. Blood culture was done in biphasic brain heart infusion medium using standard procedures. Anti-Candida IgG was detected using a commercial ELISA kit. 41.43% of HIV and 5% of cancer patients had significant levels of anti-Candida antibodies (>12 U/mL). No episode of candidemia was seen among these patients. However, one episode each of candidemia was observed among HIV and cancer patients who were negative for anti-Candida antibodies. Candidemia did not occur in any of the immunocompromised patients who had significant levels of anti-Candida antibodies. However, it is not possible to conclude from this study whether these antibodies are protective since candidemia occurred rarely in patients who were antibody negative.     

Serum Glucan Levels Are Not Specific for Presence of Fungal Infections in Intensive Care Unit Patients

Fungal infections in the critically ill patient are difficult to diagnose and are associated with a high mortality rate. A major obstacle to managing fungal infection is the lack of a reliable clinical assay that will rapidly identify patients with fungal sepsis. Glucans are polymers of glucose that are found in the cell wall of fungi and certain bacteria. Glucans are also released from the fungal cell wall into the extracellular milieu. Several studies have reported that detection of fungal glucan in serum or plasma is useful in the diagnosis of mycoses. However, recent studies have questioned the clinical utility of this assay. In this study, we examined serum glucan levels in intensive care unit (ICU) patients and attempt to correlate serum glucan levels with the presence of fungal infection. Following attainment of informed consent, serum was harvested from 46 ICU patients with confirmed fungal infections, confirmed bacterial infections, or no evidence of infection. Sera from eight healthy volunteers served as control. Serum glucan was assayed with a glucan-specific Limulus assay. Serum glucan levels were increased (69.6 ± 17 pg/ml; P < 0.001) in ICU patients versus the normal (11.5 ± 1.3 pg/ml) and noninfected ICU (27.4 ± 17 pg/ml) controls. However, serum glucan levels were not different in patients with confirmed fungal infections versus those with confirmed bacterial infections. Thus, serum glucan levels did not show a correlation with the presence of fungal infections and do not appear to be specific for fungal infections. However, the assay may be useful as a negative predictor of infection.     

Infections fongiques invasives du grand prématuré

Fungal infections were frequent in premature baby (<1500 g) and were associated with significant morbidity and mortality. In this paper we review the risk factors for invasive fungal infections and clinical settings. A better understanding of the mechanism of fungal infection in preterm infants is important in treatment and prevention. The early neonatal intensive care unit course favours colonization and proliferation of fungi since many preterm infants have central catheters and are exposed to broad spectrum antibiotics and parenteral nutrition. The majority of fungal infections in preterm neonates are due to Candida, with a small number due to other yeasts such as Malassezia. Candida is an opportunistic pathogen, which adheres to the skin, mucosal, and catheter surface. C. albicans account for 50% of cases of fungal sepsis. C. parapsilosis is the second most prevalent species in very low birth weight children; its frequency increased from 1995 to 2000. Risk factors for fungal colonization are: very low birth weight, exposure to broad spectrum antibiotics, parenteral nutrition and use of corticosteroids. Colonization of the skin, gastro-intestinal tract and respiratory tract and central vascular catheter precede infection. The majority of preterm infants with fungal infections develop thrombocytopenia, but this is a common feature shared with other sepsis. The evaluation of infants with fungal sepsis should include cerebrospinal fluid examination and culture of urine with surveillance for endocarditis, renal, liver and brain abscesses and endophthalmitis. The mortality rate can reach 30%, and is higher in very low birth weight infants.     

Serum (1-3)-β-d-Glucan as a Tool for Diagnosis of Pneumocystis jirovecii Pneumonia in Patients with Human Immunodeficiency Virus Infection or Hematological Malignancy

(1-3)-β-d-Glucan (BG) reactivity was tested in serum samples from 28 patients with human immunodeficiency virus infection or a hematological malignancy and Pneumocystis jirovecii pneumonia (PCP) and 28 control patients. The sensitivity and specificity of BG detection with the Fungitell assay for PCP were 100 and 96.4%, respectively, using a cutoff value of 100 pg/ml. Serum BG testing looks promising for the noninvasive diagnosis of PCP. Our data suggest that a higher cutoff value for the diagnosis of PCP than for the diagnosis of invasive aspergillosis or candidiasis could be used safely and will improve the specificity of the test.Pneumocystis jirovecii pneumonia (PCP) remains a serious cause of morbidity and mortality in immunocompromised patients. PCP may be difficult to diagnose owing to nonspecific signs and symptoms and possible coinfection with microorganisms other than P. jirovecii. Moreover, Pneumocystis cannot be propagated in culture. Diagnosis relies on the visualization of the fungus upon microscopic examination of induced sputum samples, bronchoalveolar lavage (BAL) fluids, or biopsy specimens. The sensitivity of microscopy varies according to the staining technique (it is highest with monoclonal antibodies) and the sample type (10). PCR detection of Pneumocystis nucleic acids has been shown to have higher sensitivity for the diagnosis of PCP than conventional staining techniques (1). However, PCR may also give positive results for patients with P. jirovecii colonization, and the clinical management of the disease in patients with positive PCR results but negative microscopy findings remains challenging. Furthermore, the diagnosis of PCP generally relies on invasive diagnostic tests, such as bronchoscopy, which is not always feasible for patients with severe respiratory distress.The measurement of serum (1-3)-β-d-glucan (BG), a cell wall component of most pathogenic fungi, including P. jirovecii, may be a useful aid for establishing the diagnosis of PCP. There are a number of diagnostic kits commercially available for detecting BG. The Fungitell BG assay (Associates of Cape Cod, East Falmouth, MA) is approved by the U.S. Food and Drug Administration as an adjunct for the diagnosis of invasive fungal disease, and the assay kit also carries the European CE mark. Up to now, data about the performance characteristics of the Fungitell BG test for the diagnosis of PCP have been scarce (2, 5, 8, 9). Few patients were included in the studies reported, and generally no relevant control patients were included. It is not known whether the cutoff value proposed by the manufacturer (80 pg/ml) can be used for the diagnosis of PCP. Elevated BG levels in PCP patients have been detected, but since many factors were reported to cause false-positive results, data for control groups are needed before the test can be used in routine practice.We retrospectively measured BG concentrations in sera from PCP patients and controls in two major risk groups, namely, patients with advanced human immunodeficiency virus (HIV) infection and patients with a hematological malignancy, with the aim of determining the diagnostic potential of BG testing for both groups.     

Animal models: an important tool in mycology.

Animal models of fungal infections are, and will remain, a key tool in the advancement of the medical mycology. Many different types of animal models of fungal infection have been developed, with murine models the most frequently used, for studies of pathogenesis, virulence, immunology, diagnosis, and therapy. The ability to control numerous variables in performing the model allows us to mimic human disease states and quantitatively monitor the course of the disease. However, no single model can answer all questions and different animal species or different routes of infection can show somewhat different results. Thus, the choice of which animal model to use must be made carefully, addressing issues of the type of human disease to mimic, the parameters to follow and collection of the appropriate data to answer those questions being asked. This review addresses a variety of uses for animal models in medical mycology. It focuses on the most clinically important diseases affecting humans and cites various examples of the different types of studies that have been performed. Overall, animal models of fungal infection will continue to be valuable tools in addressing questions concerning fungal infections and contribute to our deeper understanding of how these infections occur, progress and can be controlled and eliminated.     

Think Mycology!

Diagnosis of fungal infections demands skilled work with direct examination, fungal culture, and interpretation of macroscopic and microscopic features of the fungus as keystones of the diagnosis. We believe that medical mycology is often being treated in a stepmotherly fashion in the clinical microbiology laboratory. Drawing conclusions based on the fact that some diseases are considered never to occur in certain regions can be very dangerous. Migration, travel, possible modification of environmental circumstances and geo-ecological changes may all interfere with our endemic medical landscape. Lack of knowledge of “exotic” or “poverty-associated” diseases may lead to a significant delay in diagnosis. Nevertheless, fungal infections are emerging diseases, and lack of knowledge in the field of medical mycology is a major threat to adequate mycological diagnosis and patient management. To illustrate the importance of good mycological thinking, we present several unusual cases, all diagnosed in “ordinary” regional hospitals.     

Autopsy findings in AIDS — A histopathological analysis of fifty cases

Summary Fifty consecutive AIDS autopsy cases were evaluated. All subjects showed one or more opportunistic infections and malignancies included in the AIDS case definition with cytomegalovirus and Kaposi's sarcoma being most prevalent. Mycobacterial and cryptococcal infections occurred only infrequently. Most patients of our series after successful treatment ofPneumocystis carinii pneumonia or cerebral toxoplasmosis later succumbed to less treatable conditions like disseminated cytomegalovirus or fungal infections or malignant lymphoma. In the absence of specific treatment for the HIV infection leading to these lethal complications special emphasis must be put on the prevention of HIV transmission and spread.Abbreviations AIDS Acquired immune deficiency syndrome - CDC Centers for Disease Control - CMV Cytomegalovirus - CNS Central nervous system - HIV Human immunodeficiency virus - KS Kaposi's sarcoma - ML Malignant lymphoma - PCP Pneumocystis carinii pneumonia     

The epidemiology of febrile illness in sub-Saharan Africa: implications for diagnosis and management

BackgroundFever is among the most common symptoms of people living in Africa, and clinicians are challenged by the similar clinical features of a wide spectrum of potential aetiologies.AimTo summarize recent studies of fever aetiology in sub-Saharan Africa focusing on causes other than malaria.SourcesA narrative literature review by searching the MEDLINE database, and recent conference abstracts.ContentStudies of multiple potential causes of fever are scarce, and for many participants the infecting organism remains unidentified, or multiple co-infecting microorganisms are identified, and establishing causation is challenging. Among ambulatory patients, self-limiting arboviral infections and viral upper respiratory infections are common, occurring in up to 60% of children attending health centres. Among hospitalized patients there is a high prevalence of potentially fatal infections requiring specific treatment. Bacterial bloodstream infection and bacterial zoonoses are major causes of fever. In recent years, the prevalence of antimicrobial resistance among bacterial isolates has increased, notably with spread of extended spectrum β-lactamase-producing Enterobacteriaceae and fluoroquinolone-resistant Salmonella enterica. Among those with human immunodeficiency virus (HIV) infection, Mycobacterium tuberculosis bacteraemia has been confirmed in up to 34.8% of patients with sepsis, and fungal infections such as cryptococcosis and histoplasmosis remain important.ImplicationsUnderstanding the local epidemiology of fever aetiology, and the use of diagnostics including malaria and HIV rapid-diagnostic tests, guides healthcare workers in the management of patients with fever. Current challenges for clinicians include assessing which ambulatory patients require antibacterial drugs, and identifying hospitalized patients infected with organisms that are not susceptible to empiric antibacterial regimens.     

Intricate isavuconazole therapy of a subcutaneous nodule caused by Alternaria infectoria in a heart transplant recipient

Invasive fungal infections have appeared to be increasingly emergent in immunocompromised patients, especially in solid organ transplant (SOT) recipients. The Alternaria genus encompasses more than 80 dematiaceus species. Among them, Alternaria alternata and Alternaria infectoria are the most frequent isolated as responsible for infection in humans.To our knowledge, we report the first case of a heart transplant recipient suffering from subcutaneous nodule caused by Alternaria infectoria and who was treated with isavuconazole. Despite all the promises of this new azole drug, one should keep in mind the potential great variability of the inter-individual responses for such complex patients. We demonstrate herein how it can be challenging to manage Alternaria infection in SOT recipients. More comprehensive studies and recommendations are expected in the context of Alternaria infections.     

Emerging opportunistic fungal infections: where are we heading?

Medical mycology involves the study of pathogenic fungi and their identification in the laboratory. Mycology has developed into a field that demands the attention of all clinicians treating patients in hospitals. Interest in medical mycology has grown in recent years due to a dramatic rise in the rates of fungal infections. An overview of well-known medically significant opportunistic fungi, such as Candida, Cryptococcus, Aspergillus and Zygomycetes, as well as emerging fungal pathogens, are discussed. Antifungal failures in these individuals are high; consequently, mortality rates are also high, despite standard therapy with amphotericin-B, lipid-associated formulation of amphotericin-B and the azoles. This underscores the need for new approaches and therapies to improve outcomes in high-risk individuals.     

Techniques applicable to broncho-alveolar lavage fluid in hospital laboratory for the diagnosis of parasitic and fungal pneumopathies in immunosuppressed patients

Parasitic and fungal organisms which are likely to cause pulmonary infections in immunosuppressed patients can be detected in broncho-alveolar fluid (BAL fluid). Single and standard methods, such as direct examination of the pellet, eosine-methylene blue fast (RAL 555), cultures in usual mediums of mycology must be systematically applied to this sample and may help detect these organisms without further exploration. If the results are negative, more recent techniques can be used if they present a real asset: an easier reading and mostly an improved sensitivity. Such is the case of immuno-fluorescence assay with monoclonal antibodies for detection of Pneumocystis carinii, and inoculation of MRC5 fibroblast cell line in tissue culture for isolation of Toxoplasma. Fungal pulmonary infections diagnosis has not yet succeeded in benefiting from modern findings: latex tests proposed for the detection of circulating antigens are nor sensitive nor specific, except the "Crypto LA test". Considering the relatively frequent association with other infectious agents, the detection of a parasitic or fungal organism in the BAL fluid should not interrupt investigation of this sample; neither should it lead to hasty conclusions regarding the responsibility of this agent in acute pneumopathy. This role will have to be evaluated according to criteria which are different for each isolated organism.     

Pneumocystis carinii infection in human immunodeficiency virus-positive patients.

Pneumocystis carinii is a ubiquitous, atypical unicellular fungus. P. carinii pneumonia (PCP) is responsible for considerable morbidity and mortality in acquired immune deficiency syndrome (AIDS) patients, and is the leading complication in advanced human immunodeficiency virus (HIV) infection. Many different host (mammal)-specific species of Pneumocystis exist, but the life-cycle is not understood fully. Human strains are designated as P. carinii f. sp. (special form) hominis (at least 59 different types). P. carinii is spread via the airborne route. Disease is most frequently caused by fresh exposure to a source of P. carinii, rather than by reactivation of latent infection. Asymptomatic carriage among healthy persons may occur. PCP occurs in HIV-infected patients when the CD4+ count falls below a certain threshold; organisms multiply and gradually fill the alveoli. Symptoms, which include a mildly productive cough, progressive dyspnoea and fever, may persist for months prior to diagnosis. Without treatment, progressive respiratory insufficiency invariably ends in death. Pulmonary specimens may be obtained by procedures of varying sensitivity and risk. Diagnosis is usually confirmed by detection of stained organisms; however, staining procedures vary in sensitivity and ease of use. Robust polymerase chain reaction (PCR) protocols with good predictive results may be useful in the future. Therapy falls into two categories: for acute primary infections and for prophylaxis. A confirmed diagnosis ensures that patients do not receive potentially toxic medication (adverse drug reactions can occur). Prophylaxis can dramatically reduce the frequency of PCP in HIV patients, and its more widespread use should lead to a decline in the incidence of PCP in the future.     

Candida albicans and candidalysin in inflammatory disorders and cancer

As our understanding of mycology progresses, the impact of fungal microbes on human health has become increasingly evident. Candida albicans is a common commensal fungus that gives rise to local and systemic infections, particularly in immunocompromised patients where it can result in mortality. However, C. albicans has also been quietly linked with a variety of inflammatory disorders, to which it has traditionally been considered incidental; recent studies may now provide new aspects of these relationships for further consideration. This review provides a novel perspective on the impact of C. albicans and its peptide toxin, candidalysin, on human health, exploring their contributions to pathology within a variety of diseases.     

Effectiveness of a real-time PCR for diagnosis of Pneumocystis pneumonia in immunocompromised patients – Experience from a tertiary care center,India

Pneumocystis jirovecii pneumonia (PCP) is a life-threatening fungal infection in immunocompromised patients. Traditionally, the laboratory diagnosis of PCP relied on the visualization of organisms by microscopy as Pneumocystis cannot be readily cultured in the laboratory. The polymerase chain reaction (PCR) method is preferred over the conventional microscopic methods as PCR is rapid and found to have higher sensitivity. This retrospective study aimed to analyze the diagnostic value of a real-time PCR (qPCR) for routine diagnosis of PCP in immunocompromised patients with various underlying conditions. The qPCR targets a 121 bp fragment of P.jirovecii mitochondrial large subunit rRNA gene. The study was conducted in a 2600-bed tertiary care hospital between January and December 2019. All patients whose respiratory samples were tested for PCP by qPCR were included. The clinical diagnosis was made for each patient and categorized into PCP and non-PCP based on multi-component clinical criteria by a multi-disciplinary team. The performance characteristics of qPCR were analyzed using clinical diagnosis as the reference. A total of 339 respiratory samples from 289 patients were tested for PCP by qPCR during the study period. The overall sensitivity and specificity of qPCR were 84.75% (95% CI, 73.01% to 92.78%) and 96.1% (95% CI, 92.7 to 98.2), respectively. The sensitivity was slightly higher among HIV-infected patients (91%) than the non- HIV group (81%). The PCR exhibited higher sensitivity in bronchoalveolar lavage (BAL) (94%) than in sputum samples (81%). The colonization can be ruled out with the cycle threshold (C_T) value of below 34 with a sensitivity and specificity of 100% and 78%, respectively.The real-time PCR showed good sensitivity and specificity for routine diagnosis of PCP in patients with various underlying conditions. In addition, a cut-off C_T value (≤ 34) was determined to exclude colonization from active pneumonia.