High-throughput sequencing of cerebrospinal fluid for diagnosis of chronic Propionibacterium acnes meningitis in an allogeneic stem cell transplant recipient.

BACKGROUND: A 40-year-old man with chronic myelogenous leukemia presented multiple times over a period of 3 years with episodes of confusion, wide-based gait and falls because of recurrent hydrocephalus despite repeated therapeutic lumbar punctures. These problems occurred in the context of persistent cerebrospinal fluid (CSF) pleocytosis and leptomeningeal enhancement. Extensive diagnostic workups and therapeutic trials had failed to identify a clinically plausible cause or produce any significant improvement in the CSF and neuroimaging abnormalities. METHODS: We used high-throughput metagenomic shotgun sequencing to identify microbes in 2 CSF samples collected from the patient during his illness. These results were compared to sequence data from 1 CSF sample collected during treatment and 5 control CSF samples from other patients. RESULTS: We found sequences representing 53% and 67% of the Propionibacterium acnes genome in 2 CSF samples collected from the patient during his illness. Directed antimicrobial therapy was administered for 6 weeks with resolution of CSF and neuroimaging abnormalities. Sequencing of a sample obtained during treatment demonstrated that the P. acnes levels were decreased to background levels. After insertion of a ventriculo-peritoneal shunt, the patient returned to baseline status. CONCLUSIONS: High-throughput metagenomic shotgun sequencing revealed P. acnes as the cause of chronic meningitis that had eluded conventional attempts at diagnosis. Treatment directed at this organism resulted in cure of the infection and clinical improvement.

BK-virus and the impact of pre-emptive immunosuppression reduction: 5-year results.

A 1-year, single-center, randomized trial demonstrated that the calcineurin inhibitor or adjuvant immunosuppression, independently, does not affect BK-viruria or viremia and that monitoring and pre-emptive withdrawal of immunosuppression was associated with resolution of BK-viremia and absence of clinical BK-nephropathy without acute rejection or graft loss. A retrospective 5-year review of this trial was conducted. In cases of BK viremia, the antimetabolite was withdrawn and for sustained viremia, the calcineurin inhibitor was minimized. Five-year follow-up was available on 97% of patients. Overall 5-year patient survival was 91% and graft survival was 84%. There were no differences in patient-survival by immunosuppressive regimen or presence of BK-viremia. Immunosuppression and viremia did not influence graft survival. Acute rejection occurred in 12% by 5-years after transplant, was less common with tacrolimus versus cyclosporine (9% vs. 18%; p = 0.082), and was lowest with the tacrolimus-azathioprine regimen (5%, p = 0.127). Tacrolimus was associated with better renal function at 5-years (eGFR 63 FK vs. 52 CsA mL/min, p = 0.001). Minimization of immunosuppression upon detection of BK-viremia was associated with excellent graft survival at 5-years, low rejection rates and excellent renal function. It is a safe, short and long-term strategy that resulted in freedom from clinically evident BK-virus nephropathy.

Clinical outcomes of solid organ transplant recipients with ehrlichiosis.

Because of our experience with severe Ehrlichia infections in lung transplant recipients, we reviewed all cases of ehrlichiosis in solid organ transplant recipients at Barnes-Jewish Hospital in St. Louis, Missouri. Between 1996 and 2007, 25 cases of ehrlichiosis were identified. We retrospectively collected demographic, clinical, laboratory, and outcomes data, and we compared the 5 cases in lung transplant recipients with 20 cases in other solid organ transplant recipients (heart, 2; kidney, 13; liver, 5). The presenting symptoms in the majority of both groups consisted of fever and headache. Clinical outcomes were worse in the lung transplant group and included a greater need for intensive care unit treatment (80% vs. 20%, P=0.02), longer length of hospital stay (21 vs. 5 days, P=0.02), and propensity to develop acute lung injury or acute respiratory distress syndrome (60% vs. 10%, P=0.04). No mortalities occurred in either group of patients. In an endemic area, ehrlichiosis is not unusual in solid organ transplant recipients, and lung transplant recipients tend to have a more severe illness.

Prophylactic versus preemptive oral valganciclovir for the management of cytomegalovirus infection in adult renal transplant recipients.

Prophylaxis reduces cytomegalovirus (CMV) disease, but is associated with increased costs and risks for side effects, viral resistance and late onset CMV disease. Preemptive therapy avoids drug costs but requires frequent monitoring and may not prevent complications of asymptomatic CMV replication. Kidney transplant recipients at risk for CMV (D+/R-, D+/R+, D-/R+) were randomized to prophylaxis (valganciclovir 900 mg q.d. for 100 days, n=49) or preemptive therapy (900 mg b.i.d. for 21 days, n=49) for CMV DNAemia (CMV DNA level>2000 copies/mL in >or=1 whole blood specimens by quantitative PCR) assessed weekly for 16 weeks and at 5, 6, 9 and 12 months. More patients in the preemptive group, 29 (59%) than in the prophylaxis group, 14 (29%) developed CMV DNAemia, p=0.004. Late onset of CMV DNAemia (>100 days after transplant) occurred in 11 (24%) randomized to prophylaxis, and none randomized to preemptive therapy. Symptomatic infection occurred in five patients, four (3 D+/R- and 1 D+/R+) in the prophylactic group and one (D+/R-) in the preemptive group. Peak CMV levels were highest in the D+/R- patients. Both strategies were effective in preventing symptomatic CMV. Overall costs were similar and insensitive to wide fluctuations in costs of either monitoring or drug.

Once daily ganciclovir as initial pre-emptive therapy delayed until threshold CMV load > or =10000 copies/ml: a safe and effective strategy for allogeneic stem cell transplant patients.

Quantitative polymerase chain reaction (QPCR) for cytomegalovirus (CMV) is emerging as the preferred screening method for detection of CMV viremia in patients following allogeneic bone marrow and peripheral blood stem cell transplant. However, there are currently no universally accepted QPCR treatment thresholds at which to start pre-emptive therapy. We report here results of a pre-emptive therapy strategy using ganciclovir (GCV) 5 mg/kg initiated once daily (ODG) delayed till a threshold CMV load of > or =10 000 copies/ml whole blood in clinically stable patients. Sixty-nine at risk patients underwent allogeneic stem cell transplant. 48/69 (70%) patients had an initial episode of CMV viremia. 5/48 (10%) cleared viremia without requiring treatment. 28/43 (65%) patients requiring treatment initiated treatment with ODG. 17/28 (61%) patients successfully cleared CMV viremia on ODG, 10/28 (36%) patients required dose escalation to twice daily GCV for increasing viral loads. There were two cases of CMV disease (colitis) and no deaths due to CMV disease in patients initiating treatment with ODG. We conclude delaying pre-emptive therapy with ODG until whole blood QPCR> or =10 000 copies/ml is a safe and effective strategy for CMV viremia after allogeneic stem cell transplant in clinically stable patients.

Genotyping of Toxoplasma gondii strains from immunocompromised patients reveals high prevalence of type I strains.

Toxoplasma gondii is an important food- and waterborne opportunistic pathogen that causes severe disease in immunocompromised patients. T. gondii has an unusual clonal population structure consisting of three widespread lineages known as I, II, and III. To establish the genotypes of strains of T. gondii associated with human toxoplasmosis, we have developed a set of four highly sensitive and polymorphic nested PCR markers. Multiplex nested PCR analysis was used to genotype parasites in cerebral spinal fluid samples from 8 of 10 human immunodeficiency virus-positive patients. Remarkably, a majority of these patients had infections with type I strains or strains containing type I alleles, despite the fact that this lineage is normally uncommon in humans and animals. Multiplex analysis of these four unlinked makers was able to distinguish all three common genotypes and also detected two strains with mixed genotypes. Further analysis based on sequencing of a polymorphic intron revealed that one of these recombinant strains was an exotic lineage distinct from the archetypal clonal lineages. The multiplex nested PCR analysis described here will be useful for analyzing the contribution of parasite genotype to toxoplasmosis.

Identification of a p28 gene in Ehrlichia ewingii: evaluation of gene for use as a target for a species-specific PCR diagnostic assay.

PCR was used to amplify a 537-bp region of an Ehrlichia ewingii gene encoding a homologue of the 28-kDa major antigenic protein (P28) of Ehrlichia chaffeensis. The E. ewingii p28 gene homologue was amplified from DNA extracted from whole blood obtained from four humans and one canine with confirmed cases of infection. Sequencing of the PCR products (505 bp) revealed a partial gene with homology to outer membrane protein genes from Ehrlichia and Cowdria spp.: p30 of Ehrlichia canis (< or =71.3%), p28 of E. chaffeensis (< or =68.3%), and map1 of Cowdria ruminantium (67.3%). The peptide sequence of the E. ewingii partial gene product was deduced (168 amino acids) and the antigenicity profile was analyzed, revealing a hydrophilic protein with < or =69.1% identity to P28 of E. chaffeensis, < or =67.3% identity to P30 of E. canis, and < or =63.1% identity to MAP1 of C. ruminantium. Primers were selected from the E. ewingii p28 sequence and used to develop a species-specific PCR diagnostic assay. The p28 PCR assay amplified the expected 215-bp product from DNA that was extracted from EDTA-treated blood from each of the confirmed E. ewingii infections that were available. The assay did not produce PCR products with DNA extracted from E. chaffeensis-, E. canis-, or E. phagocytophila-infected samples, confirming the specificity of the p28 assay for E. ewingii. The sensitivity of the E. ewingii-specific PCR assay was evaluated and determined to detect as few as 38 copies of the p28 gene.

Relationship of cytomegalovirus viral load in blood to pneumonitis in lung transplant recipients.

We developed a multiplex, quantitative, real-time, polymerase chain reaction assay for cytomegalovirus (CMV) and used it to measure the CMV viral load in weekly blood specimens from 43 lung transplant recipients. The median viral load in blood samples immediately preceding bronchoscopy was 1150 copies/microg human DNA for 12 subjects with pneumonitis compared to 91 copies for 31 subjects without (P=0.02, Mann-Whitney U test). Each log10 increase in CMV viral load resulted in an increase of 1.92 in the odds ratio for CMV pneumonitis (95% confidence interval 1.03-3.56). CMV viral load was elevated (>100 copies/microg human DNA) for a median of 21 days before bronchoscopy in those subjects with pneumonitis versus 0 days in those without (P=0.004). We conclude that the risk of CMV pneumonitis after lung transplantation is related to the level of CMV DNA in blood. Quantitative PCR should be evaluated prospectively for the preemptive management of CMV in lung transplant recipients.

Infections with Ehrlichia chaffeensis and Ehrlichia ewingii in persons coinfected with human immunodeficiency virus.

The clinical course and laboratory evaluation of 21 patients coinfected with human immunodeficiency virus (HIV) and Ehrlichia chaffeensis or Ehrlichia ewingii are reviewed and summarized, including 13 cases of ehrlichiosis caused by E. chaffeensis, 4 caused by E. ewingii, and 4 caused by either E. chaffeensis or E. ewingii. Twenty patients were male, and the median CD4(+) T lymphocyte count was 137 cells/microL. Exposures to infecting ticks were linked to recreational pursuits, occupations, and peridomestic activities. For 8 patients, a diagnosis of ehrlichiosis was not considered until > or =4 days after presentation. Severe manifestations occurred more frequently among patients infected with E. chaffeensis than they did among patients infected with E. ewingii, and all 6 deaths were caused by E. chaffeensis. Ehrlichiosis may be a life-threatening illness in HIV-infected persons, and the influence of multiple factors, including recent changes in the epidemiology and medical management of HIV infection, may increase the frequency with which ehrlichioses occur in this patient cohort.

Bk virus: a clinical review.

We present a review of the clinically oriented literature about BK virus, a relative of JC virus, which is the etiologic agent of progressive multifocal leukoencephalopathy (PML). The kidney, lung, eye, liver, and brain have been proposed as sites of BK virus-associated disease, both primary and reactivated. BK virus has also been detected in tissue specimens from a variety of neoplasms. We believe that BK virus is most often permissively present in sites of disease in immunosuppressed patients, rather than being an etiologic agent that causes symptoms or pathologic findings. There is, however, strong evidence for BK virus-associated hemorrhagic cystitis and nephritis, especially in recipients of solid organ or bone marrow transplants. Now that BK virus can be identified by use of specific and sensitive techniques, careful evaluation of the clinical and pathologic presentations of patients with BK virus will allow us to form a clearer picture of viral-associated pathophysiology in many organ systems.

Kawasaki-like syndromes associated with human immunodeficiency virus infection.

Kawasaki disease is an acute febrile vasculitic syndrome of early childhood. It is very rarely seen in adults. Among the adult patients with Kawasaki disease who have been described, a disproportionate number are infected with human immunodeficiency virus (HIV). This suggests that the immunocompromised state may predispose individuals to this syndrome. We report our experience with 2 HIV-positive patients who presented with Kawasaki-like syndromes and review the published literature on HIV-positive patients with similar syndromes.

Evaluation of a commercial DNA enzyme immunoassay for detection of enterovirus reverse transcription-PCR products amplified from cerebrospinal fluid specimens.

We evaluated the DiaSorin DNA enzyme immunoassay (DEIA) kit for detection of enteroviral reverse transcription-PCR (RT-PCR) products amplified from cerebrospinal fluid. By use of an optical density of 0.05 as the absorbance cutoff, 35% of 198 specimens were PCR positive, whereas 16% were culture positive. DEIA was rapid and sensitive and can help implement enterovirus RT-PCR in clinical laboratories.

Diagnostic virology.

Diagnostic virology has now entered the mainstream of medical practice. Multiple methods are used for the laboratory diagnosis of viral infections, including viral culture, antigen detection, nucleic acid detection, and serology. The role of culture is diminishing as new immunologic and molecular tests are developed that provide more rapid results and are able to detect a larger number of viruses. This review provides specific recommendations for the diagnostic approach to clinically important viral infections.

Cerebrospinal fluid protein concentration in pediatric patients: defining clinically relevant reference values.

OBJECTIVES: To define clinically relevant reference ("normal") values for cerebrospinal fluid (CSF) protein concentrations in pediatric patients who were evaluated for meningitis by traditional criteria and by enterovirus-polymerase chain reaction (EV-PCR). DESIGN AND PATIENTS: A cohort of 906 consecutive pediatric patients to receive CSF analysis at St Louis Children's Hospital, St Louis, Mo, from June 1, 1998, to December 31,1998, was studied for clinical and laboratory data. Age-dependent CSF protein concentrations were then derived from a reference group of 225 patients in whom meningitis and other neurologic diseases were excluded by traditional clinical or laboratory criteria (excluding EV-PCR). Available CSF samples from 132 patients of the reference group were subsequently tested for EV-PCR. RESULTS: In the reference group, the CSF protein concentration was highest and most variable in neonates, with a maximum of approximately 1.0 g/L. Cerebrospinal fluid protein concentration decreased rapidly to a nadir by 6 months and remained low throughout childhood, rarely exceeding 0.3 g/L and, finally, increasing in adolescence toward adult values. Enterovirus- polymerase chain reaction was positive in CSF of 11% of the reference group, with EV-PCR-positive patients having significantly higher CSF protein concentrations than EV-PCR-negative patients aged between 4 months and 14 years. CONCLUSIONS: Reference values for CSF protein exhibit a characteristic age dependence in pediatric patients. Continued standard use of adult reference values in the pediatric population is inappropriate. The unexpected finding of a positive EV-PCR in patients not diagnosed with meningitis by traditional criteria further emphasizes the importance of selecting the most clinically relevant reference group for age and other variables when defining normal laboratory values. Arch Pediatr Adolesc Med. 2000;154:827-831

PCR amplification and phylogenetic analysis of groESL operon sequences from Ehrlichia ewingii and Ehrlichia muris.

Broad-range PCR primers were used to amplify part of the groESL operon of the canine pathogen Ehrlichia ewingii, recently recognized as a human pathogen, and the murine pathogen Ehrlichia muris. Phylogenetic analysis supported the relationships among Ehrlichia species previously determined by comparison of 16S rRNA gene sequences. These sequences provide additional PCR targets for species for which few gene sequences have been determined.

Circulation patterns of group A and B human respiratory syncytial virus genotypes in 5 communities in North America.

Human respiratory syncytial virus (HRSV) is a major cause of serious lower respiratory tract illness in infants, young children, and the elderly. To characterize the circulation patterns of HRSV strains, nucleotide sequencing of the C-terminal region of the G protein gene was performed on 34-53 isolates obtained from 5 communities during 1 epidemic year, representing distinct geographical locations in North America. Phylogenetic analysis revealed that 5-7 HRSV genotypes, including 1 or 2 predominant strains, circulated in each community. The patterns of genotypes were distinct between communities, and less diversity was seen between strains of the same genotype within than between communities. These findings are consistent with HRSV outbreaks' being community based in nature, although transmission of viruses between communities may occur. Several strains are probably introduced or circulate endemically in communities each year, and local factors-possibly immunity induced by previous years' strains-determine which strains predominate during an HRSV season.

MR imaging of herpes simplex type 1 encephalitis in infants and young children: a separate pattern of findings.

OBJECTIVE: We sought to identify the initial MR findings of herpes simplex encephalitis in infants and young children. CONCLUSION: MR imaging findings of herpes encephalitis in infants and young children appear to differ from those seen in neonates, older children, and adults. Appreciation of this MR imaging pattern coupled with a strong clinical suspicion of herpes helps to ensure the correct diagnosis is made.

The prevalence of human herpesvirus-7 in renal transplant recipients is unaffected by oral or intravenous ganciclovir.

The purpose of this study was to compare the prevalence of human herpesvirus (HHV)-7 and cytomegalovirus (CMV) viremia and the effects of oral and intravenous (iv) ganciclovir in renal transplant recipients at risk for CMV. Stored lysates from peripheral blood leukocytes from 92 patients, who had been previously analyzed for CMV viremia by polymerase chain reaction (PCR) for 12 weeks after transplantation, were analyzed for HHV-7 viremia. Baseline and peak prevalences of HHV-7 viremia were 22% and 54%, respectively (P<. 0001). Eighty-two (89%) of 92 patients had at least 1 positive PCR for HHV-7. Oral ganciclovir and treatment with iv ganciclovir had no effect on the prevalence of HHV-7 viremia. In contrast, CMV was almost completely suppressed in patients who received oral ganciclovir, and when present, CMV responded to iv therapy. These results indicate that HHV-7 is resistant to ganciclovir at levels that were effective for prevention and treatment of CMV.

Presumed parvovirus B19-associated retinal pigment epitheliopathy.

PURPOSE: To describe a patient with a white dot syndrome associated with acute erythema infectiosum. METHODS: A patient with a clinical history of erythema infectiosum and multifocal punctate white lesions at the level of the retinal pigment epithelium was followed up for an 8-month interval. Serum was tested for immunoglobulin M (IgM) and IgG antibodies to parvovirus B19 at the time of the initial evaluation and during convalescence using an indirect immunofluorescence antibody technique. Serial photographs and fluorescein angiograms were obtained. RESULTS: IgM and IgG antibodies to parvovirus were detected in the serum at the time of initial evaluation; IgM antibodies had disappeared but IgG antibodies persisted in serum obtained at 1-month follow-up. Fundus evaluation revealed clinical disappearance of some lesions, with increased pigmentation of others over the course of follow-up. CONCLUSION: The authors have identified an adult patient who presented with a white dot syndrome associated with acute erythema infectiosum documented by serologically proved parvovirus B19 infection.