Jane Kuypers

Brief Communication: Fatal Human Metapneumovirus Infection in Stem-Cell Transplant Recipients

Context Human metapneumovirus (hMPV) is a newly discovered respiratory virus. Contribution This study from a cancer referral center retrospectively examined bronchoalveolar lavage samples collected from 1995 to 1999 from hematopoietic cell transplant recipients with new or changing pulmonary infiltrates. The authors cultured hMPV in 5 of 163 symptomatic patients who were thought to have had the idiopathic pneumonia syndrome. Clinical findings included fever, cough, nasal congestion, respiratory failure, pulmonary hemorrhage, and culture-negative sepsis. Four of the patients died. Cautions All patients had moderate to severe symptoms. Implications Human metapneumovirus may cause respiratory failure in immunocompromised adults. The Editors Human metapneumovirus (hMPV) is a newly discovered respiratory virus associated with 10% to 25% of respiratory tract infections in young children (1, 2). This virus has been detected in respiratory disease at all ages (3, 4), although lower respiratory tract disease occurs more frequently in very young and very old persons and in those with underlying conditions. Clinical findings seen with hMPV infection are similar to those of respiratory syncytial virus (RSV) infection (5). As with RSV, serious disease associated with hMPV infection has been reported in immunocompromised patients. Deaths have been reported in a child (5), adults with hematologic malignant conditions (6), and adults after bone marrow or organ transplantation (7, 8). Pneumonia remains one of the most frequent serious complications of hematopoietic stem-cell transplantation (HCT) (9). Despite increased testing for pathogens in the transplantation setting, nearly 10% of myeloablative transplant recipients still develop the idiopathic pneumonia syndrome (10). We retrospectively evaluated archived bronchoalveolar lavage (BAL) fluid from patients undergoing HCT by using a sensitive hMPV-specific, real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay to determine whether hMPV may be a cause of pulmonary morbidity and mortality in this setting. Methods Patients undergoing HCT who underwent BAL between 1995 to 1999 for evaluation of respiratory disease were eligible for inclusion if they had previously consented to diagnostic studies on their tissue and if residual specimens were available and were in good condition. We retrospectively reviewed clinical and laboratory records, biopsy specimens, and autopsy specimens of all patients with detected hMPV. We also evaluated stored BAL samples in 19 HCT recipients who were enrolled in a separate prospective study evaluating BAL after transplantation. Specimens Our institution has used a diagnostic algorithm for defining the cause of pneumonia for patients with new or changing pulmonary infiltrates since 1994 (11). We processed BAL samples in a centralized laboratory for pathogens, as previously described (11). Biopsy and autopsy specimens were submitted for routine bacterial, fungal, and acid-fast bacilli culture tests; underwent cytologic examination; and were tested for mycobacteria, Legionella species, Pneumocystis jiroveci, and fungal species. Additional studies performed in real time included culture and antibody staining of BAL fluid for cytomegalovirus (CMV), RSV, parainfluenza, and influenza viruses. For our study, we assayed BAL from patients with hMPV by using PCR for RSV (12), hMPV (13), parainfluenza types 1 and 3, influenza A and B, adenovirus, human herpesvirus-6 (14), and Aspergillus species (15). In addition, we performed broad-range fungal 18S rDNA PCRs to detect other fungal pathogens (16) and a -globin PCR (17) to determine whether amplifiable DNA was present in BAL fluid. The RT-PCR assay used probes that were designed to amplify 69 and 74 base-pair fragments of the fusion protein genes from the 2 major hMPV groups (13). This assay was sensitive to 10 copies per reaction and was specific for hMPV, as previously described (18). We also examined lung tissue from autopsy specimens of patients with known RSV, CMV, and hMPV infection near the time of death after tissue digestion by using the same RT-PCR methods. Paraffin-embedded tissues were examined for the presence of hMPV antigen by immunohistochemistry in the laboratory of Dr. Thijs Kuiken, Erasmus University, Rotterdam, the Netherlands (19). We included lung sections of an experimentally infected cynomolgus monkey with hMPV as positive controls. Role of the Funding Sources The National Institutes of Health, the Fred Hutchinson Cancer Research Center, and the Adult Leukemia Research Center provided funding for this study. The funding sources had no role in the design, analysis, interpretation, or writing of this manuscript or in the decision to submit the manuscript for publication. Results Patients To determine the presence of hMPV, we evaluated BAL specimens obtained throughout the calendar year from 163 symptomatic and 19 asymptomatic HCT recipients. The mean age of patients undergoing BAL was 42 years; only 10 patients (7%) were younger than 20 years of age. Most patients (99 of 163 patients [60%]) with BAL specimens had received allogenic bone marrow transplants from unrelated donors (57 of 163 patients [35%]), matchedrelated donors (24 of 163 patients [15%]), or mismatchedrelated donors (15 of 163 patients [9%]). Another 64 patients received peripheral blood stem-cell transplants (64 of 163 patients [39%]) from matched allogenic donors (16%), autologous donors (16%), or unrelated donors (4%). One patient received an unrelated cord blood stem-cell transplant. We detected hMPV in the tested BAL specimens of 5 of 163 patients (3.0%) and in 8 of 211 specimens (3.8%). Patients were between 24 and 54 years of age, and they had received different preparative regimens and sources of stem cells (Table). We detected hMPV in samples obtained between January and April. Clinical signs and symptoms around disease onset included a low-grade fever in 4 patients, cough in 3 patients, nasal congestion in 3 patients, and sore throat in 4 patients (although transplantation-related mucositis may have confounded some symptoms). Three patients had wheezing at diagnosis. We did not note sinusitis, otalgia, otitis media, conjunctivitis, or skin rashes. All patients eventually developed radiographic evidence of diffuse pulmonary disease, and we noted rapid progression of pulmonary infiltrates in 4 of 5 patients (Figure, top). Table. Characteristics of Hematopoietic Stem-Cell Transplant Recipients with Human Metapneumovirus Infection Figure. Computed tomography scan of chest in a patient with fatal human metapneumovirus infection after transplantation ( top ) and lung histologic study in a patient with human metapneumovirus pneumonia ( bottom ). Top. Bottom. inset arrow arrow All 5 cases of hMPV lower respiratory tract disease occurred early (<40 days) after transplantation. Lymphopenia was common, although 3 patients had neutrophil engraftment at the time of hMPV detection. Diffuse alveolar hemorrhage was present in 3 patients, and prominent alveolar macrophages were present in another patient. The quantity of hMPV ranged from 2104 RNA copies/mL to 3108 RNA copies/mL of BAL fluid (Table). Detection of Potential Co-Pathogens All BAL sample test results for patients with hMPV were negative for CMV, RSV, influenza viruses, adenovirus, and fungi by real-time PCR. We did not identify any accompanying pathologic or microbiological finding in 3 patients. Herpes simplex virus was isolated at autopsy in case 3, and parainfluenza was detected by culture and PCR in case 4. Case 2 had CMV isolated from lung tissue at autopsy only. We also tested BAL samples from patients with hMPV for human herpesvirus-6. Four of eight sample test results were negative, and the remaining 3 samples had low copies of human herpesvirus-6 DNA (53, 71, and 93 human herpesvirus-6 DNA copies/mL), which are levels compatible with detection of virus in latently infected cells (14). Treatment and Outcome We treated 3 of 5 patients who received a diagnosis of diffuse alveolar hemorrhage (cases 1, 4, and 5) with high-dose corticosteroids without discernable benefit. The hMPV viral load in case 4 decreased despite the use of corticosteroids (Table). Case 2 received aerosolized ribavirin for parainfluenza pneumonia, but additional respiratory samples were unavailable for testing. Progression of respiratory symptoms was rapid, with a median of 4 days (range, 1 day to 32 days) between initiation of oxygen therapy and death. Four patients died of respiratory complications at a median of 16 days (range, 1 day to 35 days) after the BAL was found to be positive for hMPV. Three patients (cases 1, 2, and 3) also had a culture-negative sepsis syndrome that required pressor agents. Clinical diagnoses at the time of death included idiopathic pneumonia in 3 patients; sepsis, shock, or both in 3 patients; and parainfluenza pneumonia in 1 patient. The mortality rate within 3 months of the first BAL in patients with hMPV was significantly higher than that in patients in whom hMPV was not detected (Wilcoxon rank-sum test; P= 0.014). We performed a limited postmortem examination of the lungs for case 2. We noted several bilateral pleural adhesions with diffuse alveolar damage and moderate hemorrhage. The cytologic changes suggested viral infection (Figure, bottom). Postmortem lung culture grew CMV that had not been previously detected. One of 2 archived frozen tissue samples, obtained at autopsy 8 days after bronchoscopy, was found to be positive for hMPV by RT-PCR but was negative by immunohistochemistry. One HCT recipient survived hMPV infection early in her transplantation course. This young patient had neutrophil engraftment when respiratory symptoms were noted. Nonetheless, she experienced a rapidly deteriorating respiratory course and, although she was never intubated, required supplemental oxygen for 2 months. Her hoarseness, dyspnea, and cou

Genital Human Papillomavirus Infection in Women Who Have Sex with Women

Genital infection with human papillomavirus (HPV), as determined by polymerase chain reaction detection of HPV DNA and prevalence of HPV-6 and -16 serum antibodies, was investigated in 149 women who were sexually active with women. By use of HPV L1 consensus primers and hybridization to types 6/11, 16, 18, 31/33/35/39, and 45 and a generic probe, HPV DNA was detected in 30% of subjects; of these, 20% had type 31/33/35/39, 18% had type 16, and 2% had type 6/11. Of 21 subjects reporting no prior sex with men, HPV DNA was detected in 19% and squamous intraepithelial lesions in 14%. By capture ELISA with HPV-6 and -16 L1 capsids, 47% of subjects were seropositive for HPV-16 and 62% for HPV-6. Current smoking was associated with detectable HPV DNA. Genital HPV infection and squamous intraepithelial lesions are common among women who are sexually active with women and occur among those who have not had sex with men.

Seroprevalence of Human Papillomavirus Types 6 and 16 Capsid Antibodies in Homosexual Men

Human papillomavirus (HPV) has been implicated in the pathogenesis of anal carcinoma, which is increased in homosexual men. Little is known about the serologic response to HPV in normal or immunosuppressed men; therefore, HIV-infected and -uninfected homosexual men were screened for HPV-6 and -16 capsid antibodies. HIV-infected men had increased HPV DNA detection but did not significantly differ in the prevalence of serum HPV antibodies. HPV-6 DNA detection and the presence of anal warts were significantly correlated with serum antibody overall and in the HIV-infected subgroup. HPV-16 DNA detection was not significantly correlated with serum antibody overall or in either subgroup; however, HIV-infected men with high-grade anal squamous intraepithelial lesions were significantly more likely to have HPV-16 antibodies. HIV-infected men are able to generate an antibody response to HPV, and a lack of serum HPV antibodies cannot explain the increased HPV-associated disease seen in HIV-infected men.

Respiratory viruses in children with cystic fibrosis: viral detection and clinical findings

Please cite this paper as: Burns et al. (2011) Respiratory viruses in children with cystic fibrosis: viral detection and clinical findings. Influenza and Other Respiratory Viruses 6(3), 218–223.

WU and KI Polyomaviruses in Respiratory Samples from Allogeneic Hematopoietic Cell Transplant Recipients

Routine testing for these viruses in immunocompromised patients is not recommended.

Human Metapneumovirus Infections Following Hematopoietic Cell Transplantation: Factors Associated With Disease Progression

Human metapneumovirus infections among hematopoietic cell transplantation recipients correlate with infections in the community, and the risk of progression to lower respiratory tract disease is significantly increased in patients with low lymphocyte count or steroid dose of ≥1 mg/kg before diagnosis.

Comparison of the Simplexa HSV1 & 2 Direct kit and laboratory-developed real-time PCR assays for herpes simplex virus detection

BACKGROUNDnRapid detection and differentiation of herpes simplex viruses (HSV) is important for patient management and treatment, especially in HSV meningoencephalitis.nnnOBJECTIVESnResults of Simplexa HSV1 & 2 Direct kit (Focus Diagnostics), an FDA-cleared sample-to-result method providing results in ∼ 75 min, were compared to those of laboratory-developed real-time PCR assays (LDT) for detection of HSV1 and HSV2.nnnSTUDY DESIGNnSamples tested included 168 cerebral spinal fluid (CSF) collected prospectively and 150 tested retrospectively: 81 from clinical testing and 69 from subjects in a neonatal herpes study; and 53 plasma and sera. Each sample was tested by both methods on the same day.nnnRESULTSnThree of 318 CSF had invalid Simplexa Direct results and negative LDT results. Three neonatal samples with low HSV viral loads by LDT could not be typed; two were HSV2 positive and one was negative by Simplexa Direct. Of 312 CSF with valid, type-specific results, HSV1 was detected in 16 by LDT and in 17 by Simplexa Direct; HSV2 was detected in 48 by LDT and in 49 by Simplexa Direct. Concordance rates were 98.4% (κ 0.84) and 97.1% (κ 0.89) for HSV1 and HSV2, respectively. Positive percent agreements were 87.5% for HSV1 and 91.7% for HSV2. Two and four CSF were positive only by LDT and three and five were positive only by Simplexa Direct for HSV1 and HSV2, respectively.nnnCONCLUSIONSnSimplexa HSV1 & 2 assay performed well compared to an established LDT. The faster turn-around-time compared to LDT will allow for more rapid antiviral treatment and better patient management.

Applications of Digital PCR for Clinical Microbiology

ABSTRACT Digital PCR (dPCR) is an important new tool for use in the clinical microbiology laboratory. Its advantages over quantitative PCR (qPCR), including absolute quantification without a standard curve, improved precision, improved accuracy in the presence of inhibitors, and more accurate quantitation when amplification efficiency is low, make dPCR the assay of choice for several specimen testing applications. This minireview will discuss the advantages and disadvantages of dPCR compared to qPCR, its applications in clinical microbiology, and considerations for implementation of the method in a clinical laboratory.

Respiratory viruses in children with cystic fibrosis

Please cite this paper as: Burns et al. (2011) Respiratory viruses in children with cystic fibrosis: viral detection and clinical findings. Influenza and Other Respiratory Viruses 6(3), 218–223.

Respiratory virus pneumonia after hematopoietic cell transplantation (HCT)

Abstract Background. Few data exist on respiratory virus quantitation in lower respiratory samples and detection in serum from hematopoietic cell transplant (HCT) recipients with respiratory virus-associated pneumonia. Methods. We retrospectively identified HCT recipients with respiratory syncytial virus (RSV), parainfluenza virus, influenza virus, metapneumovirus (MPV), and coronavirus (CoV) detected in bronchoalveolar lavage (BAL) samples, and we tested stored BAL and/or serum samples by quantitative polymerase chain reaction. Results. In 85 BAL samples from 82 patients, median viral loads were as follows: for RSV (n=35), 2.6×106 copies/mL; for parainfluenza virus (n=35), 4.9×107 copies/mL; for influenza virus (n=9), 6.8×105 copies/mL; for MPV (n=7), 3.9×107 copies/mL; and for CoV (n=4), 1.8×105 copies/mL. Quantitative viral load was not associated with mechanical ventilation or death. Viral RNA was detected in serum samples from 6 of 66 patients: 4 of 41 with RSV pneumonia, 1 with influenza B, and 1 with MPV/influenza A virus/CoV coinfection (influenza A virus and MPV RNA detected). RSV detection in serum was associated with high viral load in BAL samples (P=.05), and viral RNA detection in serum was significantly associated with death (adjusted rate ratio, 1.8; P=.02). Conclusion. Quantitative polymerase chain reaction detects high viral loads in BAL samples from HCT recipients with respiratory virus pneumonia. Viral RNA is also detectable in the serum of patients with RSV, influenza, and MPV pneumonia and may correlate with the severity of disease.