Diagnosis of lyme disease

The use of serologic testing and its value in the diagnosis of Lyme disease remain confusing and controversial for physicians, especially concerning persons who are at low risk for the disease. The approach to diagnosing Lyme disease varies depending on the probability of disease (based on endemicity and clinical findings) and the stage at which the disease may be. In patients from endemic areas, Lyme disease may be diagnosed on clinical grounds alone in the presence of erythema migrans. These patients do not require serologic testing, although it may be considered according to patient preference. When the pretest probability is moderate (e.g., in a patient from a highly or moderately endemic area who has advanced manifestations of Lyme disease), serologic testing should be performed with the complete two-step approach in which a positive or equivocal serology is followed by a more specific Western blot test. Samples drawn from patients within four weeks of disease onset are tested by Western blot technique for both immunoglobulin M and immunoglobulin G antibodies; samples drawn more than four weeks after disease onset are tested for immunoglobulin G only. Patients who show no objective signs of Lyme disease have a low probability of the disease, and serologic testing in this group should be kept to a minimum because of the high risk of false-positive results. When unexplained non-specific systemic symptoms such as myalgia, fatigue, and paresthesias have persisted for a long time in a person from an endemic area, serologic testing should be performed with the complete two-step approach described above.     

The cost‐effectiveness of introducing nucleic acid testing to test for hepatitis B,hepatitis C,and human immunodeficiency virus among blood donors in Sweden

BACKGROUND: The purpose of this study was to estimate the cost‐effectiveness of using individual‐donor nucleic acid testing (ID‐NAT) in addition to serologic tests compared with the sole use of serologic tests for the identification of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) among blood donors in Sweden. STUDY DESIGN AND METHODS: The two strategies analyzed were serologic tests and ID‐NAT plus serologic tests. A health‐economic model was used to estimate the lifetime costs and effects. The effects were measured as infections avoided and quality‐adjusted life‐years (QALYs) gained. A societal perspective was used. RESULTS: The largest number of viral transmissions occurred with serologic testing only. However, the risks for viral transmissions were very low with both strategies. The total cost was mainly influenced by the cost of the test carried out. The cost of using ID‐NAT plus serologic tests compared to serologic tests alone was estimated at Swedish Krona (SEK) 101 million (USD 12.7 million) per avoided viral transmission. The cost per QALY gained was SEK 22 million (USD 2.7 million). CONCLUSION: Using ID‐NAT for testing against HBV, HCV, and HIV among blood donors leads to cost‐effectiveness ratios that are far beyond what is usually considered cost‐effective. The main reason for this is that with current methods, the risks for virus transmission are very low in Sweden.     

Morning admission to the hospital for surgery the same day. A practical problem for the blood bank

Morning admissions for surgery the same day are increasing because of economic incentives. These admissions permit less time for red cell serologic preparation before surgery than do the more conventional methods of patient admission. During a 4-month period, serologic problems arose in 70 of 2859 cases. In 36 of the 70 cases, the sample arrived at the blood bank about the time of the beginning of the operation; in 19 of these 36 cases, the operation had begun before serologic resolution, and in 7 of these 19, the antibody was found to be of hemolytic potential. It was concluded that administrative and logistic changes need to be made to ensure sufficient time for the serologic testing necessary for safe transfusion support for same-day surgical admissions.     

The differentiation of delayed hemolytic and delayed serologic transfusion reactions: incidence and predictors of hemolysis

BACKGROUND: After differentiation of the entities of clinically detectable delayed hemolytic (DHTR) and delayed serologic transfusion reactions (DSTR), previous investigators calculated a DHTR:DSTR incidence ratio of 18:72 from a retrospective review of patients with serologic evidence of DHTR or DSTR. There are no published data on factors that may influence the occurrence of DHTR versus DSTR in a given patient. STUDY DESIGN AND METHODS: Retrospective review was conducted of 292 patients at the Mayo Clinic who, between 1980 and 1992, received a clinical diagnosis of DHTR or DSTR concurrently with a serologic diagnosis. Red cell alloantibody specificity, the activity of the patient's reticuloendothelial system, and concurrent immunosuppression were evaluated as potential predictors of the occurrence of DHTR versus DSTR in different patients. RESULTS: The incidence of DHTR or DSTR was 1 in 1899 allogeneic red cell units transfused, with a DHTR:DSTR ratio of 36:64. Alloantibody specificity was the only variable that affected the occurrence of DHTR versus DSTR at the clinical level, with the anti-Jka and anti-Fya specificities, as well as multiple coexisting specificities, significantly associated with detectable hemolysis (p < 0.05). CONCLUSION: Clinically detectable DHTRs are found to occur more commonly than previously believed when the clinical and serologic diagnoses are made concurrently and appropriate work-ups for hemolysis are ordered. The association of certain alloantibody specificities with detectable DHTRs may have implications for clinical transfusion practice.     

Chronic mononucleosis syndrome

We present data on 14 patients with chronic symptoms of disabling fatigue in association with serologic evidence of active Epstein-Barr virus (EBV) infection. Two thirds were women, and the average age at onset was 29.6 years. Forty-three percent were known to have had previous infectious mononucleosis, but the usual criteria for that diagnosis were not helpful with the present syndrome. Eighty-six percent had serologic evidence of cytomegalovirus (CMV) infection. Profound immunodeficiency was not present, but 71% had partial hypogammaglobulinemia, and minor abnormalities of T cell subsets were noted in six of seven patients studied. Fifty-seven percent achieved temporary serologic and symptomatic remission after an average duration of 33 months. Only one patient has a sustained remission. Comparison is made with other reported chronic, recurrent, and persistent EBV syndromes, and tentative diagnostic criteria for chronic mononucleosis syndrome are presented. Recently available EBV serologic techniques allow for identification of patients who have reactivated EBV infection, and this reactivation may be related to symptoms.     

Is it Lyme disease? How to interpret results of laboratory testing.

Although laboratory testing is likely to have a greater role in diagnosis and monitoring of treatment of Lyme disease in the future, at present physicians must rely on a combination of history taking, clinical manifestations, and laboratory results. Unwarranted antibiotic therapy is to be avoided. Asymptomatic persons with positive serologic results who live in an endemic area present a challenge, and borderline serologic results in patients with manifestations of late Lyme disease are also troubling. All relevant clinical information must be considered before treatment is undertaken, until more specific and sensitive tests are available.     

Comparison of the streptozyme test with titration of antistreptolysin, antistreptokinase and antistreptohyaluronidase]

Streptozyme is a new serologic assay which is able to evidentiate 5 antistreptococcal antibodies at the same time (TAS, ASHA, ASK, DNase, NADase). This test has been carried out on 355 samples together with TAS, ASK and ASHA methods in order to value this method comparatively. The results showed that Streptozyme is a poorly reliable technique, looking at the false-positive or negative results obtained, but principally looking at the phenomena of no good reproducibility. We feel that at present it cannot be used in substitution of the classic serologic methods.     

Further aspects of microgranulocytotoxicity

Some aspects influencing the serologic outcome of complement-dependent granulocyte cytotoxicity are presented. Ficoll-Hypaque (density 1.060) gradient centrif-ugation with hypotonic lysis of red blood cells yielded populations of granulocytes with greater than 90 per cent purity. Granulocytes exposed to papain (0.01%) at 24 C for 12 minutes showed enhanced serologic reactivity compared with untreated cells. In addition, enhanced cytotoxicity was promoted by 5 C (cold) as opposed to 22 C (warm) incubation of granulocytes and antibody in the first stage of the microcytotoxicity assay and emphasizes the temperature dependence of the serologic reactions. Neutrophil-specific leukoagglutinins were not cytotoxic under optimal in vitro conditions. Conversely, a significant proportion of antibodies selected for cytotoxicity failed to agglutinate granulocytes. Cold reactive granulo-cytotoxins are complement dependent, IgM in nature, and 2-mercaptoethanol sensitive and do not appear to be immune complexes. By optimizing reaction conditions, granulocyte microcytotoxicity is a valuable addition to in vitro assays detecting immune sensitization against granulocyte surface antigens.     

Diagnosing infectious mononucleosis

Infectious mononucleosis can be diagnosed with certainty only when suggestive clinical findings are corroborated by relative and absolute lymphocytosis, lymphocyte atypia of more than 20 percent and a positive serologic test. A serologic test is not absolutely necessary when atypical lymphocytes exceed 40 percent since this is specific to infectious mononucleosis. The diagnosis is difficult when clinical findings are scanty and the blood picture is unhelpful.     

Evaluation of pregnant women exposed to respiratory viruses

Prenatal patients are often exposed to respiratory viruses at home and at work. Understandably, these patients may be concerned and want immediate answers and advice from their physicians. While most women who are exposed to chickenpox are immune, serologic testing can be performed and susceptible patients can be treated with varicella-zoster immune globulin. If the prenatal patient is infected with the varicella-zoster virus, the risk of fetal manifestations is less than 2 percent. Women who have been exposed to fifth disease can undergo serologic testing to determine the likelihood of infection. If the prenatal patient becomes infected with fifth disease during the first 20 weeks of gestation, the risk of fetal manifestations is about 9 percent and includes nonimmune hydrops and death. Cytomegalovirus, which is the most common congenital infection, is generally asymptomatic in the mother. Infected fetuses have a 25 percent chance of developing early or late neurologic manifestations. The evidence of harm from other common respiratory viruses is inconsistent.     

Development of a DNA-based genotyping method for the Diego blood group system

BACKGROUND: The paucity of appropriate reagents for serologic typing of the Diego blood group has hindered the identification of the rare Di(b-) blood donors needed to transfuse a Dib antigen-negative patient who presented with anti-Dib. Development of an alternative Di typing approach as a supplement to the current serologic typing method is an important and necessary goal. STUDY DESIGN AND METHODS: DI1 and DI2 alleles result from a single C to T substitution at nucleotide 2561 in exon 19 of the human anion exchanger gene causing a proline (DI1) to leucine (DI2) change at amino acid position 854. Allele-specific primers were designed to specifically amplify the DI1 and DI2 alleles using a PCR-based assay system. RESULTS: A PCR sequence-specific primer (SSP) method for Di genotyping was developed, and the specificity and reproducibility of the method were assessed in a blind control study using serologic tests, family segregation, and DNA sequencing analyses. A total of 1,766 DNA samples from unrelated blood donors were typed for DI1 and DI2 alleles and a single Di(b-) donor was identified. The frequency of DI1 and DI2 alleles among Chinese blood donors was 0.0357 and 0.9643, respectively. CONCLUSION: A simple, accurate, and inexpensive DNA-based PCR-SSP method was established for Di genotyping. The typing results can be visualized on a single photograph within 3 hours, making this reliable method suitable for large-scale typing of potential blood donors without serologic backup.     

How do I work up pretransfusion samples containing anti‐CD38?

Anti‐CD38 is used to treat relapsed or treatment‐refractory multiple myeloma. CD38 monoclonal antibodies, however, can interfere with routine blood bank serologic tests. Agglutination is observed at the indirect phase of testing as the drug binds to red blood cells (RBCs). Resolving the testing interference causes delays issuing RBC units to patients with anemia. A number of devised methods to eliminate or bypass the effects of anti‐CD38 on serologic tests are in use but no panacea exists. The limitations of each method require each testing site tailor an approach to best fit their needs. We present perspectives and testing practices from a hospital transfusion medicine service and an Immunohematology Reference Laboratory managing pretransfusion samples with anti‐CD38.     

Application of RHD and RHCE genotyping for correct blood group determination in chronically transfused patients

BACKGROUND: In chronically transfused patients, conventional blood group typing may be impossible because of mixed-field agglutination. STUDY DESIGN AND METHODS: In 27 patients with congenital anemia and lifelong transfusion history, genotyping for D, RHD, and RHCE was performed with polymerase chain reactions. These results were compared with the blood group typing results documented in the medical record. RESULTS: Two of 27 cases had been typed D-negative by serologic tests and D-positive by genotyping. In 20 patients, the CDE formula had been determined serologically according to the medical record; 4 of these patients were Cc by serologic tests and C/C by genotyping. One patient typed ee by serologic tests, and genotyping revealed heterozygosity (E/e). CONCLUSION: In patients with a lifelong transfusion history, serologic blood group determination may be impossible, and pretransfusion test results are not always available or reliable. In whites, Rh-matched transfusions are possible with genotyping. The genetic background of the RH genes has to be elucidated in other ethnic groups, such as in black patients with sickle cell disease, before genotyping can be applied without restriction.     

Clinical significance of RBC alloantibodies and autoantibodies in sickle cell patients who received transfusions

BACKGROUND: The clinical significance of alloimmunization to RBC antigens in sickle cell patients was analyzed by a retrospective review of the records of pediatric and adult sickle cell patients who received transfusions and who were followed over a 10-year period. STUDY DESIGN AND METHODS: Charts of pediatric and adult sickle cell patients followed at Schneider Children's Hospital (SCH) and Long Island Jewish Medical Center between 1989 and 1999 were retrieved. Patients followed at SCH were classified as pediatric, regardless of age. Data on transfusion history, alloimmunization, and transfusion reactions from 1990 were retrieved from computerized blood bank records. Transfusion history, development of alloantibodies and autoantibodies, and transfusion reactions were correlated with clinical evidence of hemolysis or other adverse reactions from the charts. All patients received ABO- and Rh-compatible blood transfusions for which a partial or extended antigen match was not performed. RESULTS: Among pediatric patients, 29 percent developed clinically significant alloantibodies, and 8 percent developed autoantibodies. Seven patients developed delayed hemolytic and/or serologic transfusion reactions, two with hyperhemolysis, two with clinical evidence of hemolysis, and three with serologic evidence only. The two patients with hyperhemolysis had received extended antigen-matched RBC transfusions to provide blood compatible with their existing antibodies. Among adult patients, 47.0 percent developed significant alloantibodies, and 9.7 percent developed autoantibodies. Five incidences of delayed hemolytic and/or serologic transfusion reactions occurred, one with hyperhemolysis and four with serologic evidence only. CONCLUSION: The alloimmunization rate is 29 percent in pediatric and 47 percent in adult sickle cell patients when partial or extended RBC antigen match is not performed. However, the delayed serologic and/or hemolytic transfusion reactions did not result in severe clinical outcome in most instances. The most important adverse event was hyperhemolysis, which may be triggered by a transfusion, but was not prevented by matching for RBC antigens. In most instances, the cause of hyperhemolysis was multifactorial.     

Koolpinyah: a virus related to kotonkan from cattle in northern Australia.

Two closely related viruses were isolated from the blood of bovines near Darwin, Northern Territory, Australia. When studies of virus morphology indicated that these were rhabdoviruses, serologic studies were done. These isolates are closely related or identical and are related to, but distinct from, the rabies-related kotonkan virus. Other serologic studies showed that these are two isolates of a newly recognized virus, for which the name Koolpinyah virus is proposed.     

Laboratory testing for suspected Lyme disease

Laboratory testing for B. burgdorferi infection is intended to substantiate a physician's clinical judgment of whether a patient has Lyme disease or not. Cultivation of B. burgdorferi from a patient's skin or blood is the gold standard for demonstration of active infection, but it is expensive and lacks clinical sensitivity. Detection of spirochetal DNA in clinical samples by PCR has better sensitivity, but PCR for B. burgdorferi has not yet been standardized for more routine diagnostic testing. Detection of antibodies to B. burgdorferi is the most practical and common approach for laboratory work-up of a case of suspected Lyme disease. Serologic assays fall short of 100% sensitivity and specificity, however, and examination of a single specimen in time does not discriminate between previous and ongoing infection. Because of a background false positivity even among healthy populations of nonendemic regions, serologic testing is recommended only when there is at least a one in five chance, in the physician's estimation, that the patient has active Lyme disease. The pretest likelihood of the disease is determined by the physician in the context of epidemiologic and clinical facts of the case. This estimate can serve to reassure patients who are at low risk of B. burgdorferi infection but are seeking a Lyme test for complaints of a more nonspecific nature. Although new subunit serologic assays based on recombinant proteins are becoming available commercially, the longstanding two-test approach, in which a positive or indeterminate result with a standardized, sensitive ELISA test is followed by verification with a more specific Western blot assay, still provides the physician with a reasonably accurate and reliable assessment of the presence of antibodies to B. burgdorferi. More recent challenges for serologic testing are seropositivity in the population as the result of immunization with the Lyme disease vaccine and the emergence of new Borrelia species that cause Lyme disease-like illnesses.     

A clinically significant erythrocyte antibody detectable only by 51Cr survival studies

Hemolytic transfusion reactions typically are explained by red cell serologic incompatibilities. We describe a patient in whom a clinically significant red cell alloantibody could not be demonstrated, despite the occurrence of several clinically severe hemolytic reactions. Serologic studies using multiple techniques demonstrated only an anti-Bga; these studies included standard procedures as well as more sensitive experimental techniques. A 51Cr survival study using red cells from a random unit, compatible in vitro with conventional techniques, showed 72 percent survival at 1 hour and 7 percent survival at 24 hours. R2R2 (hr" (e) negative) red cells in a second 51Cr survival study showed 90 percent survival at 1 hour and 92 percent survival at 6 hours. The patient was transfused with R2R2 units which were tolerated well and survived normally. Extensive serologic testing still demonstrated only an anti-Bga. A third 51Cr survival study, 10 months after the first study, with an R1R1 (hr" (e) positive) sample showed 90 percent survival at 1 hour and 42 percent survival at 6 hours. A fourth study using a larger aliquot of R2R2 (hr"(e)negative) 51Cr-labeled red cells, examined over 2 weeks showed a near normal 21-day survival of 50 percent. These 51Cr survival studies, along with normal survival of hr" (e) negative units, suggest that this patient destroys hr" (e) positive red cells despite negative serologic testing.     

Serologic distinction between human T-lymphotropic virus (HTLV) type I and HTLV type II

In November 1988, the Food and Drug Administration approved reagents for serologic screening for human T-lymphotropic virus type I (HTLV-I) infection in blood donors and patients suspected of having HTLV-I-related illnesses. These reagents are able to detect HTLV type II (HTLV-II), a close relative of HTLV-I with no known pathologic effect. The distinction between the two forms of HTLV is important to the donor and to any recipient of blood containing HTLV. The application to sera from 38 seropositive blood donors and 2 recipients (37 "confirmed" positive and 3 indeterminate by Western blot) of two methods (Western blot and peptide enzyme immunoassay) for serologic distinction between HTLV-I and -II is described. These results were compared to those from polymerase chain reaction (PCR) analysis of HTLV proviral DNA obtained from donor peripheral blood mononuclear cells. The peptide enzyme immunoassay was found to be less sensitive than the Western blot, but completely concordant with PCR results when differential reactivity could be established. The Western blot pattern showed complete diagnostic concordance with the samples with confirmed-positive serologic tests, but was incorrect in two HTLV-II-infected donors with indeterminate serologic tests. Thirty-three (89%) of the 37 individuals from this predominantly Native American and Hispanic group of blood donors were found to have HTLV-II. These findings confirm and extend previous reports that HTLV-I infection may be less common, and HTLV-II infection more common, than previously believed. The peptide enzyme immunoassay can provide most individuals who have positive results with the HTLV-I/II screening test with serologic distinction between HTLV-I and HTLV-II.     

Use of a nested polymerase chain reaction (N-PCR) to detect Trypanosoma cruzi in blood samples from chronic chagasic patients and patients with doubtful serologies

Chagas disease, caused by Trypanosoma cruzi, is an important endemic illness in Latin America. Serologic tests for T. cruzi detection in blood are sensitive, but their specificity is unsatisfactory. Direct detection of parasites in blood, either by xenodiagnosis or hemoculture, is highly specific but of low sensitivity. Molecular assays such as the Polymerase chain reaction (PCR), which amplifies certain repetitive sequences of nuclear DNA has been used as a good alternative tool for T. cruzi detection in human blood. The present study aimed to test PCR diagnosis in chagasic chronic patients and doubtful serologic patients attended in GEDOCH (Chagas Disease Study Group/UNICAMP, Brazil). A 149 bp fragment originated from nuclear DNA was specifically detected in chronic chagasic patients. The results of these tests were compared with serologic diagnosis performed using standard techniques and xenodiagnosis. We found that 43 out of 50 patients previously serodiagnosed as chagasic were positive using the N-PCR method. Thirteen of 30 patients with doubtful serologic results were confirmed as positive by N-PCR. Our results suggest that the N-PCR may be a complementary tool to serology in the diagnosis of Chagas disease, and that it is usefull for parasite detection in patients with chronic disease and patients with doubtful serologic results.     

Chagas' disease diagnosis: evaluation of several tests in blood bank screening

Blood transfusion is one of the principal routes of transmission of Chagas' disease, a major endemic disease in Latin America. Methods for blood screening are not accurate and may yield false results that lead to high social and economic costs. This study compares two methods of diagnosing Chagas' disease (indirect immunofluorescence and hemagglutination) and several enzyme-linked immunosorbent assays (ELISAs) with regard to specificity and sensitivity, by using human sera with known serologic and parasitologic characteristics, as well as samples with discrepant results on conventional serologic tests. An ELISA using recombinant antigens showed no cross-reactivity with sera that were positive for other diseases. All evaluated ELISAs performed well, and their use may lead to a reduction of more than 50 percent in the number of discordant sera. Further improvements are needed in view of the complexity of the serologic diagnosis of Chagas' disease.