Victor P. Berardi

Use of Cytomegalovirus Immune Globulin to Prevent Cytomegalovirus Disease in Renal-Transplant Recipients

We undertook a prospective randomized trial to examine whether an intravenous cytomegalovirus (CMV) immune globulin would prevent primary CMV disease in renal-transplant recipients. Fifty-nine CMV-seronegative patients who received kidneys from donors who had antibodies against CMV were assigned to receive either intravenous CMV immune globulin or no treatment. The immune globulin was administered in multiple doses over the first four months after transplantation. The incidence of virologically confirmed CMV-associated syndromes was reduced from 60 percent in controls to 21 percent in recipients of CMV immune globulin (P less than 0.01). Fungal or parasitic superinfections were not seen in globulin recipients but occurred in 20 percent of controls (P = 0.05). Only 4 percent of globulin recipients had marked leukopenia (reflecting serious CMV disease), as compared with 37 percent of the controls (P less than 0.01). There was a concomitant but not statistically significant reduction in the incidence of CMV pneumonia (17 percent of controls as compared with 4 percent of globulin recipients). A significant reduction in serious CMV-associated disease was observed even when patients were stratified according to therapy for transplant rejection (P = 0.04). We observed no effect of immune globulin on rates of viral isolation or seroconversion, suggesting that treated patients often harbored the virus but that clinically evident disease was much less likely to develop in them. We conclude that CMV immune globulin provides effective prophylaxis in renal-transplant recipients at risk for primary CMV disease.

Seroprevalence of human immunodeficiency virus among childbearing women: estimation by testing samples of blood from newborns

Attempts to predict the course of the epidemic of acquired immunodeficiency syndrome (AIDS) have been hampered by the lack of an objective, practical way to estimate the prevalence of infection with the human immunodeficiency virus (HIV) in the general population. Testing for the prevalence of HIV infection in women should be a sensitive means to track the epidemic and to study the potential for perinatal transmission. Antibodies in maternal blood are contained in neonatal blood specimens routinely collected on absorbent paper for other purposes, such as screening for phenylketonuria; we therefore tested for HIV antibody in these specimens. Analysis of batches of individually blinded specimens from selected hospitals protected the anonymity of the mothers and babies and was cost efficient. Using the newborns blood as an indicator of the mothers serologic status, we concluded that 1 of every 476 women (2.1 per 1000) giving birth in Massachusetts was positive for HIV antibody by immunofluorescence assay or enzyme-linked immunosorbent assay, both confirmed by immunoblot (Western blot) testing. The prevalence of HIV infection varied according to the type and location of the maternity hospitals; rates of seropositivity were highest in inner-city hospitals (8.0 per 1000), lower in mixed urban and suburban hospitals (2.5 per 1000), and lowest in suburban and rural hospitals (0.9 per 1000). This method is useful for collecting data needed to plan and evaluate prevention strategies and to predict the health care resources that will be needed to care for women and children who contract AIDS. Because other states have newborn screening programs similar to the Massachusetts program, this approach can be used for national surveillance of AIDS in women.

The long-term clinical outcomes of Lyme disease. A population-based retrospective cohort study.

Lyme borreliosis is a tick-borne infection caused by the spirochete Borrelia burgdorferi [1-3]. The disease usually begins with erythema migrans accompanied by viral-like or meningitis-like symptoms. Weeks later meningitis, facial palsy, atrioventricular nodal block, or migratory musculoskeletal pain may develop, followed months to years later by episodes of frank arthritis, encephalopathy, polyneuropathy, or acrodermatitis [4]. Lyme disease is now the most common vector-borne disease in the United States; nearly 50 000 patients have been diagnosed with it since 1982 [5]. Musculoskeletal and neurologic sequelae may occur from Lyme disease. Some of the late consequences of Lyme disease, such as oligoarticular arthritis, axonal polyneuropathy, or active encephalopathy, are thought to be caused by persistent spirochetal infection and are amenable to antibiotic treatment [6-8]. Other syndromes such as persistent arthritis, fibromyalgia, subtle joint pain, or mild encephalopathy do not improve with antibiotic treatment, suggesting a mechanism other than active infection [9-12]. We studied persons residing in an endemic coastal area of Massachusetts who were previously infected with B. burgdorferi in the early 1980s [13]. They contracted Lyme disease while the clinical syndromes and optimal antibiotic therapies were still evolving, which offered a natural experiment for the identification of risk factors for Lyme disease sequelae. We ascertained the prevalence of persistent symptoms in unselected patients with a history of Lyme disease; ascertained their rheumatologic, neurologic, and health status outcomes; and identified potential risk factors for these long-term sequelae. Methods Participants In February l991, we did a follow-up analysis of residents of Argylla Road in Ipswich, Massachusetts, an endemic coastal area for Lyme disease. The incidence and clinical course of Lyme disease among residents of this area have been reported previously [13]. Participants were recruited by calling consecutive households located in the Argylla Road area, the epicenter of infection, to ask if they would be interested in enrolling in a study about Lyme disease in their area. Potential participants were told that the study involved a history, physical examination, and serologic analysis for Lyme disease. Information about whether a person ever had a previous diagnosis of Lyme disease was obtained and used to assign tentative status (with or without Lyme disease) for study participants. We recruited participants until we had 50 tentative persons with Lyme disease and 50 tentative controls. Once the potential Lyme disease group was filled, calls were made consecutively to fill the potential control group. Residents 18 years of age or older were invited to participate in the study. This protocol was approved by the Brigham & Womens Hospital Committee for the Protection of Human Subjects. Confirmation of Lyme Disease For inclusion in the Lyme group, persons needed a previous diagnosis of Lyme disease by a physician and needed to fulfill the Centers for Disease Control and Prevention (CDC) criteria for Lyme disease (a history of physician-documented erythema migrans or a late manifestation of Lyme disease confirmed by a positive Lyme serologic test result, or both [14]). This information was obtained through patient interview and then medical record review to determine if patients fulfilled criteria for Lyme disease. Previous study records, local physician reports, and previous serologic test results were available for confirmation of Lyme disease. Persons without a previous clinical history of Lyme disease were classified as controls. The status of the participants (with or without Lyme disease) was determined independent of the clinical assessment, using a protocol that did not include any outcome data. Assessment of Clinical Outcomes A blinded investigator determined outcomes in a standardized manner independent of Lyme disease status. All patients completed a standardized questionnaire, had electrocardiography, and had a neuropsychological battery of tests. The questionnaire included data on demographics, comorbidity, education, review of systems, medications, memory and cognitive function, and the Short Form-36 health status measure (a reliable, previously validated measure of physical, psychological, social, and role functions [15]). A physical examination was done by one observer blinded to Lyme disease status. It included a joint examination (the American College of Rheumatology Glossary examination) that measured swelling and pain through passive range of motion [16] and a neurologic evaluation of strength and deep tendon reflexes, light touch, and vibration sensation with a 128-Hz tuning fork (at the elbow, wrist, fibula, and ankles). Pain and swelling indices from the joint examination (the American College of Rheumatology Glossary examination) were summed and recorded as a global score. A vibration test result of a distal gradient was considered present if the participant reported diminished vibratory sensation at a distal compared with proximal site. Each participant had an electrocardiographic study that was interpreted blindly by a cardiologist uninvolved with the clinical assessment. All outcomes were determined by one investigator who had no knowledge of whether participants were in the Lyme or control groups. The neuropsychological battery of tests measured immediate and delayed verbal memory, attention, conceptualization, fine motor dexterity, and perceptual discrimination. Tests included the California Verbal Learning Test [17], Wechsler Memory Scale (visual reproduction and verbal paired associates subtests [18]), Shipley abstraction subtest [19], Stroop test [20], Trailmaking test [21], and Purdue Pegboard Test [22]. The California Verbal Learning Test measures verbal memory. Participants are asked to learn a list of 16 words during five trials; recall on the fifth trial is recorded (trial 5). This is then followed by a distracter list. The original list is recalled after the distracter list is learned (short recall) and then recalled again after a 20-minute delay (long recall). This is a challenging test of memory for patients with superior premorbid experience. Normative values are available for young and elderly adults [17]; the range of normal is between 11 and 15 words for trial 5 and is between 10 and 15 words for the long-recall subtest for persons between 45 and 54 years of age. A clinically significant change in the California Verbal Learning Test would be recalling 4 more words or 4 fewer words. All tests were administered according to published procedures. Test scores were transformed into standard scores calculated from published, age-corrected normative data. Participants with a score of 2 or more SDs from age-adjusted means were considered impaired. All results were reviewed by a neuropsychologist (RK) who was not involved with the participants evaluation, to determine those patients who were in need of further clinical evaluation. Participants with swelling or pain (joint examination test result), evidence of a distal gradient (vibration test result) or persistent symptoms of paresthesias in an extremity, or impairment on two or more neurocognitive tests were sent for further clinical evaluation. Nine patients were evaluated at the Lyme disease clinic at Tufts-New England Medical Center and 4 were evaluated by other neurologist or rheumatologist consultants to determine if these abnormal screening test results were accompanied by objective findings. This evaluation included lumbar puncture, electrophysiologic studies, magnetic resonance imaging, detailed neuropsychological tests [8], joint radiographs, or arthrocentesis. Serologic Evaluation All patients had serologic testing after the history and examination. Serum samples were stored at 70C and were tested for IgG antibodies to B. burgdorferi by indirect enzyme-linked immunosorbent assay (ELISA [23]); for IgM, IgG, and IgA antibodies to the spirochete by antibody-capture enzyme immunoassays [24]; and for the pattern of IgG reactivity to spirochetal polypeptides by Western blotting. In general, Western blot reactivity varied with the degree and duration of dissemination of Lyme disease. For example, patients with early localized infection or erythema migrans might react to only 2 to 8 B. burgdorferi polypeptides, those with meningitis might react to at least 8 to 14 polypeptides, and those with arthritis or late central nervous system disease might react to as many as 18 to 25 polypeptides (Berardi VP. Personal communication). The isolate used for antigen preparations was the B. burgdorferi G39/40 strain obtained through low passage [24]. Indirect ELISA titers greater than 400 and ELISA capture ratios (sample optical density/control optical density) of 1.0 or more were considered as increased test results. Western blot reactivity to five or more B. burgdorferi-specific polypeptides indicated previous infection [25]. Silver Stain Method The Dieterle silver impregnation stain used was a modification made by one of us (PHD) in 1985 [26]. This standard approach has yielded a constant clean yellow background of cerebral cortex sections with no silver impregnation of anatomic neural processes and dendrites. Spirochetes are easily seen as black to blue-black cells against the yellow tissue. Specificity for nonstaining of normal tissue fibers (procollagen, elastin, basement membrane material, and neural dendrites and filaments) and documentation of the cytologic structure of Borrelia spirochete strains were further tested in a large extended study [27]. Controls routinely used in each stain assay consisted of NP40 strain that was injected into human normal breast tissue removed for cosmetic surgery and was paraffin-embedded in the usual manner (negative control), and rat gonad tissue infected with the Reiter strain of treponemal spirochetes (positive c

Meningoencephalitis from Borrelia miyamotoi in an Immunocompromised Patient

Ixodes ticks serve as vectors for Borrelia burgdorferi, the agent of Lyme disease. Globally, these ticks often concurrently harbor B. miyamotoi, a spirochete that is classified within the relapsing-fever group of spirochetes. Although humans presumably are exposed to B. miyamotoi, there are limited data suggesting disease attributable to it. We report a case of progressive mental deterioration in an older, immunocompromised patient, and even though Kochs postulates were not met, we posit B. miyamotoi as the cause, owing to its direct detection in cerebrospinal fluid (CSF) with the use of microscopy and a polymerase-chain-reaction (PCR) assay. It is likely that B. miyamotoi is an underrecognized cause of disease, especially in sites where Lyme disease is endemic.

Borrelia burgdorferi in Joint Fluid in Chronic Lyme Arthritis

Although indirect evidence suggests that chronic Lyme arthritis is caused by persistent infection with Borrelia burgdorferi, direct visualization has been lacking. We report the demonstration of B. burgdorferi from synovial fluid aspirated from the right knee of a 31-year-old man with Lyme arthritis for more than 1 year. After 6 days, culture medium inoculated with synovial fluid showed one motile and several nonmotile spirochetes. Direct immunofluorescence staining showed reactivity with anti-B. burgdorferi serum. Spirochetes were not seen in subcultured material. The patients arthritis improved with high-dose intravenous penicillin. Identification of B. burgdorferi from the joint fluid of a patient with long-standing arthritis supports the concept that the arthritis is due to persistent infection.

Rapid emergence of a focal epidemic of Lyme disease in coastal Massachusetts

We describe a focal epidemic of Lyme disease, which spread from a nature preserve and affected an adjacent community of permanent residents in coastal Massachusetts. The attack rate from 1980 through 1987 was 35 percent among 190 residents living within 5 km of the nature preserve and was greatest (66 percent) among those living closest to the preserve. The risk of infection bore little relation to sex or age. Late Lyme disease, which clustered near the preserve, occurred mainly in residents infected early in the epidemic who did not have a history of erythema migrans and did not receive antibiotic therapy. All the residents with serologic evidence of infection had early or late clinical manifestations of Lyme disease, or both, during the period of study. The seasonal risk of infection was bimodal--greatest in June, with a secondary peak in October--and corresponded to periods of increased transmission. In the nature preserve, the density of the vector tick, Ixodes dammini, exceeded that in other New England sites. The zoonosis rapidly became endemic, and the severity of its impact correlated with the abundance of deer. This epidemic of Lyme disease demonstrated that outbreaks can be focal and can spread rapidly within a community of permanent residents.

Borrelia miyamotoi Disease in the Northeastern United States: A Case Series

Context The first known cases of Borrelia miyamotoi infection in North America were reported in 2013. The incidence, prevalence, and clinical spectrum of this infection are currently undefined. Contribution A review of the clinical records of 51 of 97 patients with acute B. miyamotoi infection diagnosed by polymerase chain reaction testing at a reference laboratory provides preliminary data on the clinical presentation, course, and response to antibiotics of this infection. Caution The study was not prospective or population-based. Implication Borrelia miyamotoi disease may be an emerging tickborne infection of clinical significance in the northeastern United States. The public health burden of infectious agents transmitted by the deer tick (Ixodes dammini, also known as the blacklegged tick or I. scapularis), comprising Borrelia burgdorferi, Babesia microti, Anaplasma phagocytophilum, and deer tick virus (Powassan virus lineage II), seems to be increasing in the northeastern United States (14). The first human cases of infection with Borrelia miyamotoi, previously considered nonpathogenic, were identified in Russia and described in 2011 (5, 6). In 2013, the American index case of B. miyamotoi disease (BMD) was described (7), and 2 other case patients (8) were reported with a clinical illness that was initially diagnosed as human anaplasmosis. Cases have subsequently been identified in New England (9), the Netherlands (10), and Japan (11). The prevalence of B. miyamotoi in host-seeking deer ticks (about 1% to 5% [1214]) is such that human exposure is likely; indeed, a serosurvey suggests that 10% of tick-exposed New England residents may have been exposed to B. miyamotoi (15). It is possible that zoonotic transmission has been common but not discriminated from other deer ticktransmitted zoonoses because the acute clinical presentation of BMD cases reported to date is nonspecific. To better define the clinical spectrum of this newly recognized zoonosis, we identified definitive cases of acute BMD by polymerase chain reaction (PCR) analysis of patient blood samples sent to a reference laboratory by clinical practices. We summarized the clinical features of BMD from 51 case patients out of 97 whom we identified as having active B. miyamotoi infection. In addition, we describe relevant associated laboratory findings and compare the observed frequency of BMD with that of human anaplasmosis and babesiosis, thereby providing a preliminary assessment of its relative public health burden. Methods Blood Samples Emergency departments, urgent care clinics, and primary care offices in Massachusetts, Rhode Island, New Jersey, and New York sent whole blood samples (EDTA-anticoagulated) to IMUGEN (Norwood, Massachusetts) as part of the clinical management of patients who were acutely symptomatic with features or laboratory findings suggestive of possible tickborne infection (typically fever, myalgia, flu-like illness, headache, or rash). An active detection protocol was adopted to identify samples that contained evidence of B. miyamotoi DNA during the 2013 and 2014 transmission seasons (1 April to 30 November). A positive B. miyamotoi finding on PCR prompted a request for chart review, and we focused on patients under our care with a goal of obtaining detailed clinical histories on at least 50 patients. The study was approved by the New England Institutional Review Board, and written, informed consent was obtained for clinical and laboratory follow-up of patients with positive PCR findings for Borrelia species. Molecular Detection of Infection Specimen receipt and handling, DNA extraction, and PCR setup were performed with enhanced contamination control practices and precautions at IMUGEN, which operates under good laboratory practice standards, Clinical Laboratory Improvement Amendments and College of American Pathologists certification, and New York State approval (Clinical Laboratory Evaluation Program). The laboratory has environmentally separate specimen processing, archive, and molecular extraction and setup areas. Extracted DNA from whole blood was tested in triplicate for the presence of Borrelia species by real-time PCR (Appendix) (16). Real-time PCR targeting the msp2 gene of A. phagocytophilum and the 18S ribosomal RNA gene of B. microti was performed from whole blood DNA extractions as previously described (1720). Representative samples were independently analyzed by PCR sequencing at the Telford laboratory of Tufts University (Appendix). B. burgdorferi Serology Sera were tested at IMUGEN by antibody capture enzyme immunoassay (EIA) for IgM, IgA, and IgG isotypes to B. burgdorferi sensu stricto strain 49736 and by immunoblot for IgM and IgG isotypes to B. burgdorferi sensu stricto strain G39/40 (2123). Immunoblot findings were interpreted according to the 2-tiered protocol (24). Recombinant B. miyamotoi GlpQ Enzyme-Linked Immunosorbent Assay The glycerophosphodiester phosphodiesterase (GlpQ) antigen has been found to be useful in the serodiagnosis of relapsing fever (25, 26). Given the genetic relatedness of B. miyamotoi to relapsing fever spirochetes, we analyzed the utility of GlpQ for confirmation of a BMD diagnosis. The GlpQ gene sequence from B. miyamotoi (GenBank accession number AY368276) was used as the basis for cloning and expression as a 38-kDa recombinant protein (rGlpQ), which was used in an indirect EIA for detection of antibody to B. miyamotoi (Appendix). Case Definitions A positive blood sample on 2 independent PCR assays (genus- and species-specific) was considered definitive evidence of active B. burgdorferi or B. miyamotoi infection. A blood sample that was repeatedly positive (reextracted and retested from the original sample) for B. microti or A. phagocytophilum on PCR was considered definitive evidence of active infection. Role of the Funding Source Internal funding from IMUGEN enabled the development and validation of test methods, the study design and execution, the clinical analysis, and the laboratory studies. The decision to prepare a manuscript for publication was not predicated on the availability of funding or the funding source. Results Confirmation of B. miyamotoi as the Infecting Agent We attempted to sequence portions of the GlpQ and flagellin genes, as previously described (7), from the 23 BMD cases from 2013 for which we had adequate remaining blood samples for DNA extraction. The organism was confirmed to be B. miyamotoi (GenBank accession numbers KP754938 to KP754960) in 17 samples (GlpQ, fla, or both sequences were obtained from the sample). The other 6 samples contained few spirochetes; those with high cycle threshold values in the real-time GlpQ PCR did not provide sequence (Spearman rank correlation coefficient, 0.67 [P< 0.001]). Frequency of BMD Between 1 April 2013 and 31 October 2014, we engaged in active case detection using specimens submitted to IMUGEN and identified 97 patients whose samples contained B. miyamotoi DNA. Of these, we obtained clinical information on 51 who had not been previously reported (7, 8) from selected practices in Massachusetts, Rhode Island, New Jersey, and New York. The months of onset or diagnosis of these BMD cases (Figure 1) overlapped with those for the other deer ticktransmitted infections, although most cases had onset in July and August. Figure 1. Seasonal distribution of acute Borrelia miyamotoi incident infections. The months of acute blood collection are presented for 97 samples submitted in 20132014 that were determined by active case detection or retrospectively to contain B. miyamotoi DNA. To provide a crude estimate of the incidence of BMD, we compared the frequency of PCR-confirmed findings of B. burgdorferi, B. miyamotoi, B. microti, and A. phagocytophilum in the patient samples submitted during the peak transmission months (May through October) in 2013 and 2014. Of 11515 unique samples that were submitted and tested for all 4 agents, 3.1% (exact binomial 95% CI, 2.7% to 3.4%) contained B. microti DNA, 1.4% (CI, 1.2% to 1.6%) contained A. phagocytophilum DNA, and 2.5% (CI, 2.2% to 2.8%) contained Borrelia species DNA. Borrelia miyamotoi DNA was detected in 0.8% (CI, 0.6% to 1.0%) of all samples; hence, 1.7% (CI, 1.5% to 1.9%) of all samples contained B. burgdorferi or cognate sequences that were not assigned to species. Clinical Spectrum of BMD Basic clinical data were available for 51 case patients with definitive BMD selected out of 97 total. The clinical spectrum of disease was variable, but presenting symptoms were often suggestive of an undifferentiated flu-like illness. Patients presented with acute headache, fever, and chills and were often found to have leukopenia, thrombocytopenia, and elevated aminotransferase levels, mimicking human anaplasmosis infection (Tables 1 and 2). Patients were commonly described as appearing toxic; more than 50% were suspected of having sepsis, and 24% required hospitalization. The headaches were most commonly described as severe, resulting in head computed tomography scans and spinal taps in 5 patients. Two patients presented with recurrent fever, and 1 of them, who was not treated initially, yielded blood samples drawn a month apart that were positive on PCR. Table 1. Clinical Features of the 51 Case Patients With BMD Table 2. Laboratory Findings for the 51 Case Patients With BMD Treatment regimens were typical for suspected tickborne disease in our referral area practices. Of the 51 case patients, 40 received a 2- to 4-week course of oral doxycycline. Seven of 51 received oral amoxicillin, and 3 of these had received 1 or 2 doses of ceftriaxone beforehand. One of 51 received levofloxacin for 10 days. One case patient first received atovaquone and azithromycin for a laboratory-confirmed diagnosis of babesiosis and later received ceftriaxone after the results of laboratory assays were reported. Of the 42 patients for whom information on outcome was available, 40 had prompt resolution of

Eastern equine encephalitis in Massachusetts A report of 16 cases, 1970–1984

We studied the case records of 16 patients with eastern equine encephalitis (EEE) in Massachusetts from 1970 to 1984 and compared their presentations, courses, and outcomes with the data available from previous epidemics. In recent years, there has been a greater frequency of EEE in adults, whereas in the past it was considered a disease of children. Also, prognosis for a good functional recovery seems to be correlated with age over 40 years, a long prodromal course (5 to 7 days) of constitutional symptoms, and the absence of coma. Previous reports did not mention these significant correlations. We also stress the positive and negative diagnostic correlations, in order to distinguish between EEE and herpes simplex encephalitis.

Coexposure to Borrelia burgdorferi and Babesia microti Does Not Worsen the Long-Term Outcome of Lyme Disease

Previous studies suggest that concurrent Lyme disease and babesiosis produce a more sever illness than either disease alone. The majority of babesiosis infections, however, are subclinical. Our objective was to characterize on the basis of a total-population survey of Nantucket Island, Massachusetts, whether coexposure to Lyme disease and babesiosis causes more severe illness or poorer long-term outcomes than Lyme disease alone. In this retrospective cohort study, residents indicating a history of Lyme disease were compared with randomly selected population controls on a standardized medical history, blinded physical examination, and serological studies for Borrelia burgdorferi and Babesia microti. Serological evidence of exposure to babesiosis was not associated with increased severity of acute Lyme disease. The groups did not differ with regard to the prevalence of constitutional, musculoskeletal, or neurological symptoms a mean of 6 years after acute Lyme disease. Prior Lyme disease and serological exposure to B. microti are not associated with poorer long-term outcomes or more persistent symptoms Lyme disease alone.

Investigational screening for Babesia microti in a large repository of blood donor samples from nonendemic and endemic areas of the United States.

Babesia microti, a transfusion‐transmissible intraerythrocytic parasite, is increasing in frequency in the United States with no available FDA‐licensed donor screening assay. We utilized investigational arrayed fluorescence immunoassay (AFIA) and polymerase chain reaction (PCR) to detect B. microti antibodies and DNA in blood donors.