Invasive fusariosis in patients with leukaemia in the era of mould-active azoles: increasing incidence, frequent breakthrough infections and lack of improved outcomes.

OBJECTIVES: Historically, patients with leukaemia and invasive fusariosis (IF) have experienced poor outcomes in the setting of persistent immunosuppression. Herein, we retrospectively reviewed the incidence, presentation and outcomes of IF that are scarcely studied in contemporary cohorts of leukaemia patients. METHODS: We identified adult leukaemia patients with proven or probable IF at MD Anderson Cancer Center during 2009-21. Independent risk factors for 42 day mortality after IF diagnosis were determined using a multivariable logistic regression model. Combined with historical data, the annual IF incidence density over the past 23 years was estimated using Poisson regression analysis. RESULTS: Among 140 leukaemia patients with IF (114 proven), 118 patients (84%) had relapsed/refractory leukaemia and 124 (89%) had neutropenia at IF diagnosis. One hundred patients (71%) had pulmonary IF, 88 (63%) had disseminated IF and 48 (34%) had fungaemia. Coinfections were common (55%). Eighty-nine patients (64%) had breakthrough IF to mould-active triazoles. Most patients (84%) received combination antifungal therapy. Neutrophil recovery [adjusted OR (aOR), 0.04; 95% CI, 0.01-0.14; P < 0.0001], pulmonary IF (aOR, 3.28; 95% CI, 1.11-9.70; P = 0.032) and high SOFA score (aOR, 1.91 per 1-point increase; 95% CI, 1.47-2.50; P < 0.0001) were independent predictors of 42 day mortality outcomes. From 1998 to 2021, IF incidence density increased significantly at an annual ratio of 1.03 (95% CI, 1.01-1.06; P = 0.04). CONCLUSIONS: IF is predominantly seen in patients with relapsed/refractory leukaemia and increasingly seen as a breakthrough infection to mould-active triazoles. Despite frequent combination antifungal therapy, high mortality rates have persisted in patients with lasting neutropenia.

Immunocompromised Host Pneumonia: Definitions and Diagnostic Criteria: An Official American Thoracic Society Workshop Report.

Pneumonia imposes a significant clinical burden on people with immunocompromising conditions. Millions of individuals live with compromised immunity because of cytotoxic cancer treatments, biological therapies, organ transplants, inherited and acquired immunodeficiencies, and other immune disorders. Despite broad awareness among clinicians that these patients are at increased risk for developing infectious pneumonia, immunocompromised people are often excluded from pneumonia clinical guidelines and treatment trials. The absence of a widely accepted definition for immunocompromised host pneumonia is a significant knowledge gap that hampers consistent clinical care and research for infectious pneumonia in these vulnerable populations. To address this gap, the American Thoracic Society convened a workshop whose participants had expertise in pulmonary disease, infectious diseases, immunology, genetics, and laboratory medicine, with the goal of defining the entity of immunocompromised host pneumonia and its diagnostic criteria.

Blockade of the PD-1/PD-L1 Immune Checkpoint Pathway Improves Infection Outcomes and Enhances Fungicidal Host Defense in a Murine Model of Invasive Pulmonary Mucormycosis.

Anecdotal clinical reports suggested a benefit of adjunct immune checkpoint inhibitors (ICIs) to treat invasive mucormycosis. However, proof-of-concept data in animal models and mechanistic insights into the effects of ICIs on host defense against Mucorales are lacking. Therefore, we studied the effects of PD-1 and PD-L1 inhibitors (4 doses of 250 µg/kg) on outcomes and immunopathology of invasive pulmonary mucormycosis (IPM) in cyclophosphamide- and cortisone acetate-immunosuppressed mice. Rhizopus arrhizus-infected mice receiving either of the ICI treatments had significantly improved survival, less morbidity, and lower fungal burden compared to isotype-treated infected mice. While early improvement of morbidity/mortality was comparable between the ICI treatments, anti-PD-L1 provided more consistent sustained protection through day 7 post-infection than anti-PD-1. Both ICIs enhanced the fungicidal activity of ex-vivo splenocytes and effectively counteracted T-cell exhaustion; however, macrophages of ICI-treated mice showed compensatory upregulation of other checkpoint markers. Anti-PD-1 elicited stronger pulmonary release of proinflammatory cytokines and chemokines than anti-PD-L1, but also induced cytokines associated with potentially unfavorable type 2 T-helper-cell and regulatory T-cell responses. Although no signs of hyperinflammatory toxicity were observed, mice with IPM receiving ICIs, particularly anti-PD-1, had elevated serum levels of IL-6, a cytokine linked to ICI toxicities. Altogether, inhibition of the PD-1/PD-L1 pathway improved clinical outcomes of IPM in immunosuppressed mice, even without concomitant antifungals. PD-L1 inhibition yielded more favorable immune responses and more consistent protection from IPM-associated morbidity and mortality than PD-1 blockade. Future dose-effect studies are needed to define the "sweet spot" between ICI-induced augmentation of antifungal immunity and potential immunotoxicities.

Invasive mould infections in patients from floodwater-damaged areas after hurricane Harvey - a closer look at an immunocompromised cancer patient population.

OBJECTIVES: Extensive floodwater damage following hurricane Harvey raised concerns of increase in invasive mould infections (IMIs), especially in immunocompromised patients. To more comprehensively characterize the IMI landscape pre- and post-Harvey, we used a modified, less restrictive clinical IMI (mcIMI) definition by incorporating therapeutic-intent antifungal drug prescriptions combined with an expanded list of host and clinical features. METHODS: We reviewed 103 patients at MD Anderson Cancer Center (Houston, Texas), who lived in Harvey-affected counties and had mould-positive cultures within 12 months pre-/post-Harvey (36 and 67 patients, respectively). Cases were classified as proven or probable IMI (EORTC/MSG criteria), mcIMI, or colonization/contamination. We also compared in-hospital mortality and 42- day survival outcomes of patients with mcIMI pre-/post-Harvey. RESULTS: The number of patients with mould- positive cultures from Harvey-affected counties almost doubled from 36 pre- Harvey to 67 post- Harvey (p < 0.01). In contrast, no significant changes in (mc)IMI incidence post-Harvey nor changes in the aetiological mould genera were noted. However, patients with mcIMIs from flood affected areas had significantly higher in-hospital mortality (p = 0.01). CONCLUSIONS: We observed increased colonization but no excess cases of (mc)IMIs in immunosuppressed cancer patients from affected areas following a large flooding event such as hurricane Harvey.

Cat Scratch Disease as a Mimicker of Malignancy.

Cat scratch disease (CSD) infrequently mimics malignancy. We reviewed 11 such cases at MD Anderson Cancer Center and an additional 36 reported from the literature. Breast cancer, sarcoma, and lymphoma were the most commonly suspected malignancies. Most patients were young, female, had prior cat exposure, and had no systemic symptoms. Regional lymphadenopathy was the most common finding.

Clinical presentation and outcomes of non-typhoidal Salmonella infections in patients with cancer.

BACKGROUND: Non-typhoidal Salmonella (NTS) infection is thought to be more severe in cancer patients, but this has not been studied since the development of new cancer therapies, increasing antibiotic resistance and the introduction of new antibiotics. We sought to describe the demographic characteristics, microbiological findings, clinical manifestations, and outcomes of NTS infections in cancer patients at our institution. METHODS: We reviewed microbiology laboratory records and identified patients who had cancer and from whom NTS organisms were recovered between January 1, 2000 and December 31, 2013, at a comprehensive cancer center in Houston, Texas. Descriptive statistics were used to summarize patient characteristics, clinical presentation and outcomes. RESULTS: We identified 110 isolates from 82 patients with 88 episodes of NTS infection (including five relapses [6%] in four patients, and two consecutive episodes in one patient). Fifty-five patients (67%) had hematologic malignancies. Most NTS isolates were susceptible to the commonly prescribed antimicrobials. Sixty-nine percent of patients had sepsis and one-third had severe sepsis or septic shock. Gastroenteritis, bacteremia, or both were present in 69% of patients, and the rest had focal infection. Mortality at 30 days was low (8%). Relapses occurred only in patients receiving ≤ 10 days of antibiotic therapy. CONCLUSIONS: NTS affects predominantly patients with hematologic malignancies, followed by gastrointestinal and genitourinary cancers. Invasive disease, sepsis, and septic shock are common presentations among admitted patients. Antimicrobial prophylaxis may not prevent NTS infection. Thirty-day mortality and attributable mortality rates were low in our series compared to older case series. Early appropriate antibiotic therapy may have had a role in decreasing mortality. Relapses occurred in patients receiving ≤ 10 days of therapy, suggesting the need for longer duration of antibiotic therapy in cancer patients with uncomplicated NTS infections.

Breakthrough Mucormycosis Developing on Mucorales-Active Antifungals Portrays a Poor Prognosis in Patients with Hematologic Cancer.

Although breakthrough mucormycosis (BT-MCR) is known to develop on mold-active antifungals without Mucorales activity, it can also occur while on Mucorales-active antifungals. Herein, we retrospectively compared the characteristics and outcomes of patients with hematologic malignancies (HMs) or hematopoietic stem cell transplant (HSCT) who developed BT-MCR on mold-active antifungals with or without Mucorales activity. Of the patients developing BT-MCR, 16 were on Mucorales-active antifungals (9 isavuconazole, 6 posaconazole, 1 amphotericin B), and 87 were on other mold-active agents (52 voriconazole, 22 echinocandins, 8 itraconazole, 5 echinocandin + voriconazole). Both groups were largely comparable in clinical characteristics. Patients developing BT-MCR while on Mucorales-active antifungals had higher 42-day mortality, from either symptom onset (63% versus 25%, p = 0.006) or treatment initiation (69% versus 39%, p = 0.028). In multivariate Cox regression analysis, exposure to Mucorales-active antifungals prior to BT-MCR had a hazard ratio of 2.40 (p = 0.015) for 42-day mortality from treatment initiation and 4.63 (p < 0.001) for 42-day mortality from symptom onset. Intensive care unit (ICU) admission and APACHE II score at diagnosis, non-recovered severe neutropenia, active HM, and amphotericin B/caspofungin combination treatment were additional independent predictors of 42-day mortality. In summary, BT-MCR on Mucorales-active antifungals portrays poor prognosis in HM/HSCT patients. Moreover, improvements in early diagnosis and treatment are urgently needed in these patients.

Development and validation of a risk assessment tool for BKPyV Replication in allogeneic stem cell transplant recipients.

BACKGROUND: BK polymavirus (BKPyV), a member of the family Polyomaviridae, is associated with increased morbidity and mortality in allogeneic stem cell transplant recipients. METHODS: In our previous retrospective study of 2477 stem cell transplant patients, BKPyV replication independently predicted chronic kidney disease and poor survival. In this study, using the same cohort, we derived and validated a risk grading system to identify patients at risk of BKPyV replication after transplantation in a user-friendly modality. We used 3 baseline variables (conditioning regimen, HLA match status, and underlying cancer diagnosis) that significantly predicted BKPyV replication in our initial study in a subdistribution hazard model with death as a competing risk. We also developed a nomogram of the hazard model as a visual aid. The AUC of the ROC of the risk-score-only model was 0.65. We further stratified the patients on the basis of risk score into low-, moderate-, and high-risk groups. RESULTS: The total risk score was significantly associated with BKPyV replication (P < .0001). At 30 days after transplantation, the low-risk (score ≤ 0) patients had a 9% chance of developing symptomatic BKPyV replication, while the high-risk (score ≥ 8) of the population had 56% of developing BKPyV replication. We validated the risk score using a separate cohort of 1478 patients. The AUC of the ROC of the risk-score-only model was 0.59. Both the total risk score and 3-level risk variable were significantly associated with BKPyV replication in this cohort (P < .0001). CONCLUSIONS: This grading system for the risk of symptomatic BKPyV replication may help in early monitoring and intervention to prevent BKPyV-associated morbidity, mortality, and kidney function decline.

Protective Activity of Programmed Cell Death Protein 1 Blockade and Synergy With Caspofungin in a Murine Invasive Pulmonary Aspergillosis Model.

Pharmacological immune checkpoint blockade has revolutionized oncological therapies, and its remarkable success has sparked interest in expanding checkpoint inhibitor therapy in infectious diseases. Herein, we evaluated the efficacy of programmed cell death protein 1 (PD-1) blockade in a murine invasive pulmonary aspergillosis model. We found that, compared with isotype-treated infected control mice, anti-PD-1-treated mice had improved survival, reduced fungal burden, increased lung concentrations of proinflammatory cytokines and neutrophil-attracting chemokines, and enhanced pulmonary leukocyte accumulation. Furthermore, combined treatment with anti-PD-1 and caspofungin resulted in a significant survival benefit compared with caspofungin or anti-PD-1 therapy alone, indicating a synergistic effect between PD-1 inhibitors and immunomodulatory antifungal agents.

Corrigendum: Outcomes in Invasive Pulmonary Aspergillosis Infections Complicated by Respiratory Viral Infections in Patients With Hematologic Malignancies: A Case-Control Study.

[This corrects the article DOI: 10.1093/ofid/ofz247.].

Outcomes in Invasive Pulmonary Aspergillosis Infections Complicated by Respiratory Viral Infections in Patients With Hematologic Malignancies: A Case-Control Study.

BACKGROUND: Data regarding invasive pulmonary aspergillosis (IPA) following respiratory viral infections (RVIs) in patients with leukemia and/or hematopoietic stem cell transplantation (LHSCT) are limited. METHODS: We conducted a retrospective case-control study of post-RVI IPA (2006-2016). Cases were patients who underwent LHSCT and had RVI due to respiratory syncytial virus (RSV), influenza virus (INF), or parainfluenza virus (PIV) followed by culture-documented IPA within 6 weeks. Controls had IPA only. RESULTS: We identified 54 cases and 142 controls. Among cases, 29 (54%) had PIV infection, 14 (26%) had INF infection, and 11 (20%) had RSV infection. The median time to IPA after RVI was 7 days. A greater percentage of cases (37 [69%]) than controls (52 [37%]) underwent allogeneic HSCT (P < .0001). Cases were more likely to be nonneutropenic (33 [61%] vs 56 [39%]; P = .009) and in hematologic remission (27 [50%] vs 39 [27%]; P = .003) before IPA. Cases were more likely to have monocytopenia (45 [83%] vs 99 [70%]; P = .05) and less likely to have severe neutropenia (21 [39%] vs 86 [61%]; P = .007) at IPA diagnosis. Prior use of an Aspergillus-active triazole was more common in cases (27 of 28 [96%] vs 50 of 74 [68%]; P = .0017). Median time to empirical antifungal therapy initiation was 2 days in both groups. Crude 42-day mortality rates did not differ between cases (22%) and controls (27%), but the 42-day mortality rate was higher among cases with IPA after RSV infection (45%) than among those with IPA following INF or PIV infection (13%; P = .05). CONCLUSIONS: IPA had comparable outcomes when it followed RVI in patients who underwent LHSCT, and post-RVI IPA occurred more frequently in patients with prior allogeneic HSCT and was associated with leukemia relapse and neutropenia.

Infections with the agent of 'kennel cough' in patients with cancer.

OBJECTIVE: To investigate the clinical manifestations, microbiological data, and outcomes of Bordetella bronchiseptica (Bb) infections in patients with cancer. METHODS: Review of electronic medical records of 24 patients with Bb infection, from 2000 to 2013. An infection was considered to be associated with Bb if both clinical manifestations plus microbial growth from infected sites were present. RESULTS: Ten patients (42%) had a monomicrobial infection, whereas multiple pathogens in addition to Bb were isolated from the rest (14 patients, 58%). The most frequent sites of infection were the respiratory tract (18 patients, 75 %) and bloodstream (17%). The most frequently associated conditions were lymphopenia (71%), tobacco use (42%), and chemotherapeutic or immunosuppressive agents (33% each). Animal exposure was established in four patients. Overall, the response rate to treatment was 100% for monomicrobial and 79% for polymicrobial infections, respectively. CONCLUSIONS: Bb is an uncommon pathogen even in immunosuppressed patients. Predominant sites of infection are the respiratory tract and bloodstream. Bb should be considered pathogenic in immunocompromised hosts, particularly with history of zoonotic exposure, even if accompanied by co-pathogens. Therefore, contact with potential animal sources should be minimized. The infection ranges from mild to severe and has no specific clinical or radiographic manifestations.

Carbapenem versus Cefepime or Piperacillin-Tazobactam for Empiric Treatment of Bacteremia Due to Extended-Spectrum-ß-Lactamase-Producing Escherichia coli in Patients with Hematologic Malignancy.

Infections with extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli are common in patients with hematologic malignancy. The utility of cefepime and piperacillin-tazobactam as empiric therapy for ESBL-producing E. coli bacteremia in patients with hematologic malignancy is largely unknown. We conducted a single-center, retrospective cohort review of 103 adult inpatients with leukemia and/or hematopoietic stem cell transplant (HCT) recipients with monomicrobial ESBL-producing E. coli bacteremia. No association between increased 14-day mortality and empiric treatment with cefepime (8%) or piperacillin-tazobactam (0%) relative to that with carbapenems (19%) was observed (P = 0.19 and P = 0.04, respectively). This observation was consistent in multivariate Cox proportional hazards models adjusted for confounding and an inverse probability of treatment-weighted (IPTW) Cox proportional hazards model. Both fever and persistent bacteremia were more common in patients treated empirically with cefepime or piperacillin-tazobactam. Empiric treatment with cefepime or piperacillin-tazobactam did not result in increased mortality relative to that with treatment with carbapenems in patients with hematologic malignancy and ESBL-producing E. coli bacteremia, although most patients were changed to carbapenems early in treatment. However, due to prolonged fever and persistent bacteremia, their role may be limited in this patient population.

Clinical Outcomes Associated With Linezolid Resistance in Leukemia Patients With Linezolid-Resistant Staphylococcus epidermidis Bacteremia.

BACKGROUND: Coagulase-negative staphylococci, including Staphylococcus epidermidis, are the most common cause of bloodstream infection in cancer patients. Linezolid resistance is increasingly identified in S. epidermidis, but whether such resistance alters the clinical course of S. epidermidis infections is unknown. The purpose of this study was to assess the clinical impact of linezolid resistance in leukemia patients with S. epidermidis bloodstream infection. METHODS: This was a retrospective, single-center cohort study of all adult leukemia patients with S. epidermidis bacteremia treated with empiric linezolid between 2012 and 2015. The primary end point was adverse clinical outcome on day 3, defined as a composite of persistent bacteremia, fever, intensive care unit admission, or death. Fourteen- and 30-day mortality were also assessed. RESULTS: Eighty-two unique leukemia patients with S. epidermidis were identified. Linezolid resistance was identified in 33/82 (40%). Patients with linezolid-resistant S. epidermidis were significantly more likely to have persistent bacteremia (41% vs 7%; adjusted relative risk [aRR], 5.15; 95% confidence interval [CI], 1.63-16.30; P = .005); however, adverse short-term clinical outcomes overall were not more common among patients with linezolid-resistant S. epidermidis (61% vs 33%; aRR, 1.46; 95% CI, 0.92-2.32; P = .108). No differences were observed in 14- or 30-day mortality. CONCLUSIONS: Leukemia patients with linezolid-resistant S. epidermidis bacteremia who were treated with linezolid were significantly more likely to have persistent bacteremia compared with those with linezolid-sensitive isolates. Interventions to limit the clinical impact of linezolid-resistant S. epidermidis are warranted.

Mixed mold pulmonary infections in haematological cancer patients in a tertiary care cancer centre.

There is a paucity of data regarding mixed mold pulmonary infections (MMPIs) in patients with haematological malignancies with or without haematopoietic stem cell transplantation (HSCT). We retrospectively studied 27 such patients (2005-2015) and compared them to patients with invasive pulmonary aspergillosis (IPA) caused by Aspergillus fumigatus. Factors associated with the diagnosis of MMPIs were significant corticosteroid use [20 (74%) vs 6 (22%), P < 0.001], sputum as the source specimen [13 (48%) vs 3 (11%), P = 0.003], younger age (median age: 58 vs 66 years, P = 0.006), and male sex [22 (81%) vs 13 (48%), P = 0.01]. Haematological cancers other than acute myeloid leukaemia (AML)/myelodysplastic syndromes (MDS) were less common in MMPIs than in IPA patients [AML/MDS: 6 (22%) vs 14 (52%), P = 0.04]. Only significant corticosteroid use [95% CI (2.7-42.7), P < 0.001], and sputum as the source specimen [95% (1.6-41.6), P = 0.012] were statistically significant as independently associated with increased risk of MMPIs diagnosis in multivariate analysis. Total mortality rate at day 42 postdiagnosis was comparable in both groups.

Fulminant Cryptococcus neoformans infection with fatal pericardial tamponade in a patient with chronic myelomonocytic leukaemia who was treated with ruxolitinib: Case report and review of fungal pericarditis.

Cryptococcus neoformans is a saprophytic fungal pathogen that can cause serious illness in immune-compromised hosts and it presents with a wide variety of clinical symptoms. We present a fatal case of fulminant C. neoformans infection presenting as pericardial tamponade in a 71-year-old male with chronic myelomonocytic leukaemia undergoing chemotherapy with the JAK-STAT inhibitor ruxolitinib. We also review the published cases of fungal pericarditis/tamponade. In addition to illustrating an atypical presentation of C. neoformans, this case highlights the risk for opportunistic fungal infections in patients with haematological malignancies, especially the ones treated with small molecule kinase inhibitors.

Invasive mold infections of the central nervous system in patients with hematologic cancer or stem cell transplantation (2000-2016): Uncommon, with improved survival but still deadly often.

BACKGROUND: Historically considered to have very poor outcome, there is paucity of recent data regarding invasive mold infections (IMIs) of the central nervous system (CNS) in patients with hematologic cancer (HC) or stem cell transplantation (SCT). METHODS: We reviewed the records of HC patients and/or SCT recipients who were diagnosed with CNS IMIs (EORTC/MSG criteria) at MD Anderson Cancer Center (1/1/2000-6/31/2016). Risk factors for survival at day (d) 42 post diagnosis were assessed. RESULTS: We identified 40 such patients (16 with proven infection). The incidence density was 3.8 cases/100000 patient days and mortality remained stable throughout the study period. Most patients had active HC and neutropenia at diagnosis (95% and 53% respectively). Of the 25 patients with a microbiological diagnosis, Aspergillus spp and Mucorales accounted for 85% of cases. CNS IMIs were deemed to be secondary to hematogenous spread in 31 (77%), mostly (90%) fungal pneumonia. CNS lesions typically presented as solitary ring-enhancing abscesses in MRI (26; 65%). Most patients (34; 85%) received lipid AMB and were treated with combination therapy (33; 83%); Mortality 42d was 48%. In univariate analysis, lack of surgical drainage (p = 0.01), absence of giant cells (p = 0.01) and granulomas (p = 0.03) were associated with increased 42d mortality. In multivariate analysis, co-infection was associated with increased (p = 0.005), while steroid tapering was associated with decreased mortality (p = 0.01). CONCLUSIONS: Although less lethal, improved outcome in these uncommon infections was related only to immune response in histopathology, steroid tapering and possibly surgical drainage. In a contemporary 16-year cohort of 40 patients with hematologic cancer and mold infections of Central Nervous System, 42-day mortality was 48%. Improved survival was related to immune response in histopathology, absence of co-infections, corticosteroid tapering and possibly surgical drainage.

Initial Treatment of Cancer Patients with Fluconazole-Susceptible Dose-Dependent Candida glabrata Fungemia: Better Outcome with an Echinocandin or Polyene Compared to an Azole?

The 28-day crude mortality rate in 68 cancer patients with fluconazole-susceptible dose-dependent Candida glabrata fungemia started on treatment (within 48 h after blood culture collection) with an echinocandin or liposomal amphotericin-B was better (30%) than those treated with azole monotherapy (52%) (P = 0.07). After adjusting for confounders, azole monotherapy also was associated with worse 28-day survival (hazard ratio, 3.8; P = 0.003).

Plasma Viral miRNAs Indicate a High Prevalence of Occult Viral Infections.

Prevalence of Kaposi sarcoma-associated herpesvirus (KSHV/HHV-8) varies greatly in different populations. We hypothesized that the actual prevalence of KSHV/HHV8 infection in humans is underestimated by the currently available serological tests. We analyzed four independent patient cohorts with post-surgical or post-chemotherapy sepsis, chronic lymphocytic leukemia and post-surgical patients with abdominal surgical interventions. Levels of specific KSHV-encoded miRNAs were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and KSHV/HHV-8 IgG were measured by immunoassay. We also measured specific miRNAs from Epstein Barr Virus (EBV), a virus closely related to KSHV/HHV-8, and determined the EBV serological status by ELISA for Epstein-Barr nuclear antigen 1 (EBNA-1) IgG. Finally, we identified the viral miRNAs by in situ hybridization (ISH) in bone marrow cells. In training/validation settings using independent multi-institutional cohorts of 300 plasma samples, we identified in 78.50% of the samples detectable expression of at least one of the three tested KSHV-miRNAs by RT-qPCR, while only 27.57% of samples were found to be seropositive for KSHV/HHV-8 IgG (P<0.001). The prevalence of KSHV infection based on miRNAs qPCR is significantly higher than the prevalence determined by seropositivity, and this is more obvious for immuno-depressed patients. Plasma viral miRNAs quantification proved that EBV infection is ubiquitous. Measurement of viral miRNAs by qPCR has the potential to become the "gold" standard method to detect certain viral infections in clinical practice.