Polymerase Chain Reaction In Peripheral Blood Research Articles

Infecção experimental por Trypanosoma vivax em caprinos no semiárido brasileiro: detecção de DNA de Trypanosoma vivax no colostro e avaliação da transmissão lactogênica

ABSTRACT: This study aimed to identify the presence of Trypanosoma vivax DNA in the colostrum of infected goats and to explore the possibility of transmission for neonates fed using colostrum collected from infected goats. We used twelve goats in the final third of gestation with an age of approximately 24 months. Six goats were inoculated intravenously with 0.5mL of blood containing approximately 1.25x105 trypomastigotes of T. vivax, and six remained uninfected. The presence of T. vivax in colostrum was evaluated by Polymerase Chain Reaction (PCR). The possibility of T. vivax transmission by colostrum was assessed by feeding six neonates born of serologically negative goats using colostrum from infected goats. Peripheral blood from neonates was collected daily for thirty days to assess the T. vivax presence through the examination of Giemsa-stained smears of leukocyte layers with the buffy coat technique (BCT) and by PCR. The results of a direct examination of colostrum were negative, but PCR confirmed the presence of T. vivax DNA in all infected goats. Additionally, lactogenic transmission by colostrum was not demonstrated once both BCT and PCR of neonate peripheral blood were negative.

Chronic myeloid leukaemia with pulmonary leucostasis in a young Down syndrome patient

We reported a rare case of chronic myeloid leukaemia (CML) complicated with pulmonary leucostasis in an 11-year-old Down syndrome (DS) patient who presented with respiratory distress. His peripheral blood investigation showed features of CML with 17% circulating blast, with immunophenotyping showing positivity towards myeloid markers. Peripheral blood polymerase chain reaction for BCR–ABL fusion transcripts was positive, and cytogenetic studies showed Ph chromosome with trisomy 21. Despite cytoreduction therapy with hydroxyurea and leukapheresis, the patient succumbed due to shock, with multiple organ failure. Our case highlights the need for early detection and rapid referral for aggressive treatment in DS patients with CML, as the combination is associated with a poor outcome.

Impact of Post-Transplant High-Dose Cyclophosphamide and Donor Sources on CMV Reactivation.

Introduction Cytomegalovirus (CMV) reactivation remains a common cause of morbidity and mortality following allogeneic hematopoietic cell transplant (HCT) despite attempts at prophylaxis. Following transplantation, surveillance of the peripheral blood CMV viral load with early aggressive treatment is commonly employed and has been an effective strategy at preventing overt infection. While a matched related donor (MRD) is often the preferred transplant type, the use of alternative donors including matched unrelated donors (MUD), mismatched unrelated donors (MMUD) and haploidentical (HI) transplants, has greatly increased over the past several years. The data regarding CMV infection among these alternative donor types is scarce. Objectives To better characterize the extent and risk factors for CMV reactivation among patients who received an allogeneic HCT for hematologic malignancies, particularly among HI transplant recipients and those who received post-transplant high-dose cyclophosphamide (PTCy) for graft-versus-host disease (GVHD) prophylaxis. Methods A retrospective analysis was performed on 88 adults with hematologic malignancies who received an allogeneic HCT at our institution from 2012-2017. Only cases with CMV seropositive donors and/or recipients were included. We compared donor types/sources, conditioning regimens, GVHD prophylaxis regimens, and assessed for CMV reactivation utilizing the peripheral blood polymerase chain reaction (PCR) values that are monitored in post-transplant clinic visits. Results Nineteen (22%) patients received MRD transplants, while the remainder received alternative donor sources, including 17 (19%) HI transplants. Overall, the rate of CMV reactivation was 27% with a significant difference noted between donor sources (p= Conclusion HI HCT transplant recipients are at high risk for CMV reactivation. The use of myeloablative conditioning regimens or PTCy did not affect the rate of CMV reactivation.

PCR sensitivity of peripheral blood of dogs co-infected with Leishmania spp. and Ehrlichia spp. in endemic area of Brazil.

Peripheral blood of 400 dogs infected with Leishmania and Ehrlichia were analyzed using polymerase chain reaction (PCR), and clinical signs were characterized. PCR and parasitological tests were conducted. PCR was positive for Leishmania in 84.75%, and parasitological tests showed that 63.25% and 31.75% were positive for Leishmania and Ehrlichia, respectively. All animals showed more than three clinical signs. PCR results were negative for Leishmania in 15.25% of the samples. Conventional PCR of peripheral blood can be used for diagnosing canine visceral leishmaniasis in combination with other techniques, especially in uncertain cases that need species identification.

Herpes simplex virus-1 and cytomegalovirus DNAs detection in the inner ear of implanted patients with non-congenital infection

Conclusions: The detection of cytomegalovirus (CMV) and herpes simplex virus-1 (HSV-1) genome in perilymph of patients with negative serology or clinical history for congenital infections supports the hypothesis that Herpesviridae, even after acquired postnatal infections, could remain in latent phase in the spiral ganglion and damage the cochlea by a possible subsequent reactivation. Further studies are needed to identify the markers of such reactivation.Objective: To identify the presence of certain viral species in the endolabyrinthic fluid of deaf patients with non-congenital infection. The research of viral DNA within the inner ear is the only direct way to increase our knowledge about the viral role in postnatal damage to the cochlea.Methods: Thirty-six patients (1–69 years) suffering from bilateral sensorineural hearing loss (SNHL) were subjected, during cochlear implant (CI) surgery, to a sample taking of inner ear fluid. Several types of viral genome (HSV, VZV, CMV, EBV and Enterovirus) were investigated in each sample through multiplex polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR). Radiological exams, serology (specific IgG and IgM) and PCR of peripheral blood were also performed.Results: While the research of the viral genome in peripheral blood was negative in all patients, multiplex PCR on endolabyrinthic fluid samples was positive in three patients (two cases of CMV-DNA and one case of HSV-1 DNA).

Comparative Diagnostic Utility of IS6110 PCR Assay in CSF and Peripheral Blood Samples of Tuberculous Meningitis Patients: A Pilot Study from Central India.

Tuberculous Meningitis (TBM) is the most severe form of Central Nervous System Tuberculosis (CNS-TB) and constitutes about 6% of all Extrapulmonary TB (EPTB) cases. Most guidelines for the diagnosis and management of TBM agree on the use of simple Cerebrospinal Fluid (CSF) analysis using molecular tools like Polymerase Chain Reaction (PCR). However, the sensitivity of PCR varies while using a CSF sample. In the present study, we have compared the diagnostic utility of PCR assay in both peripheral blood and CSF sample collected from TBM cases. To evaluate the application of the peripheral blood PCR assay as an alternate tool for TBM diagnosis compared to conventional CSF-PCR based system. A total of 50 TBM patients were prospectively recruited from in patient department wards of Central India Institute of Medical Sciences (CIIMS) between January 2014 - Feburary 2015. Mycobacterium tuberculosis (MTB) specific IS6110 PCR and BactT liquid culture were performed in 20 of recruited cases classified as Stage 1, 2 and 3 based on British Medical Research Council (BMRC) contemporary clinical criteria for the severity of TBM. Clinical characteristics were summarised in terms of percentages for categorical variables, i.e., age groups, gender, signs and symptoms. All statistical analysis was carried out using MedCalc software version 11.6. Overall IS6110 PCR positivity in CSF was around 80% (16/20), which was higher than culture (29.3%) and peripheral blood (39%). Out of 8 positive cases, stage wise positivity of peripheral blood PCR assay in three TBM stages was 0% (stage1) 50% (stage 2) and 67% (Stage 3) respectively. Positivity of peripheral blood PCR was significantly more (86%) in patients with CSF culture/ IS6110 PCR positive for MTB infection with sensitivity and specificity of 50 and 100% respectively. Increased positivity rates of peripheral blood PCR was observed with decreased CSF/Blood sugar ratio in stage 3 cases, suggesting enhanced probability of mycobacterial blood dissemination in cases of TBM severity. Our results suggest that although the molecular diagnosis of TBM infection in CSF remains the method of choice, peripheral blood based PCR can be used as a good alternative to CSF in case of TBM severity where the repeated CSF collection may be needed. However, study demands further validation in large cohorts to justify the present hypothesis.

Toxoplasmose ocular com reação em cadeia da polimerase positiva em sangue periférico – relato de dois casos, estado de São Paulo, Brasil

Aims: To describe the use of polymerase chain reaction (PCR) in peripheral blood and demonstrate its importance in the clinical follow-up of patients with ocular toxoplasmosis. Case description: Two immunocompetent patients were clinically diagnosed with acute ocular toxoplasmosis. The routine clinical evaluation consisted of fundus examination using binocular indirect ophthalmoscopy, color fundus photography, fluorescein angiography, and spectral domain optical coherence tomography. The serological diagnosis was made by ELISA (IgM, IgG) and confirmed by ELFA (IgG, IgM). The molecular diagnosis was made by PCR in peripheral blood using the B1 gene of Toxoplasma gondii as marker. The younger patient was male, had previous lesion in the right eye, complained of low visual acuity in the left eye and was under treatment. The older patient was male, had retinal detachment, and presented with sudden loss of acuity in the right eye. The fundus examination revealed chorioretinal scar in the left eye. IgG was reactive, IgM was non-reactive, and PCR was positive in the peripheral blood of both patients. New blood samples were collected for serological and molecular monitoring and PCR remained positive in both cases. Six weeks after treatment with oral sulfadiazine and pyrimethamine, the PCR yielded negative results. Conclusions: The results show that T. gondii antigens may be found in peripheral blood during ocular reactivations and that PCR may be a good tool for the follow-up of patients with ocular toxoplasmosis.

Allogeneic Stem Cell Transplantation Using In Vivo T-Cell Depletion in Myeloid Disorders

Introduction:Alemtuzumab is used for in vivo T-cell depletion to reduce graft versus host disease in allogeneic Stem Cell Transplantation (SCT). Profound lymhotoxicity of this monocloncal antibody can potentially increase morbidity and mortality in SCT due to excessive viral infections and increased risk of graft rejection. We retrospectively analysed outcome of patients with myeloid disorders (acute myeloid leukaemia and myelodysplasia) who received in vivo T-cell depletion using Alemtuzumab based conditioning for allogeneic SCT over a period of 3 years in our centre.Methods:Patients were identified from department transplant data base. Data was collected for 73 consecutive patients over a period of three years using patient medical records, clinical work station and electronic patient records. The conditioning regimen included Fludarabine 30mg/m2 x5 (days -7 to -3), Alemtuzumab 10mgx5 (days -8 to -4) and Melphalan 140mg/m2 (day -2). Chimerism analysis was performed by polymerase chain reaction (PCR) to identify short tandem repeats within peripheral blood leucocytes and CD3 fraction. The quantification of donor chimerism was done by using gel photography system and LabWroks software. Viral testing was performed by PCR analysis.Results:Median age for the patients was 59 years (range 41-71). Median duration of follow up was 19 months (4-49 months). The majority of patients (67%) received SCT from a voluntary unrelated donor. Sixty four patients (88%) had AML whilst 9 had myelodysplasia. Sixty five (89%) patients were in morphological complete remission (<5% blasts) at the time of SCT. Median HCT-Comorbidity index was 2 (range 1-5). Median time to neutrophil engraftment was 13 days (range 9-23) and platelet engraftment 14 days (median 8-48). During the course of transplantation, 50 patients were treated for neutropenic fever with broad spectrum antibiotics and 16 for presumed fungal infection. Forty three patients (59%) reactivated cytomegalovirus (CMV), 10 (13%) had Ebstein Barr Virus (EBV) viremia and 4 (5%) were found to have adenovirus on peripheral blood PCR analysis after SCT. Denovo acute graft versus host disease (grade 2-4) required treatment in 12 patients (16%) whilst chronic GVHD was seen in 14% (n=10). 20 patients required donor lymphocyte infusion due to mixed chimerism after SCT. Total incidence of acute GVHD (grade 2-4) was 23% (n=17) and that of chronic GVHD was 15%. Median donor chimerism in peripheral blood leucocyte fraction was 100% at day 30, 60, 90 and 180 after SCT. Chimerism analysis on CD3 compartment showed 100%, 96%, 95.5% and 95% donor fraction respectively at these time points. Predicted overall survival at 2 years was 53% and Event Free Survival 49%. Non relapsed mortality in this cohort was 18% at one year. One patient had primary graft rejection with no secondary graft rejections. Overall survival was not statistically different between those who were treated for viremia (CMV, EBV, adenovirus) when compared to those who did not have viremia (p=0.31). No deaths were attributed to either CMV or EBV.Conclusion:Our retrospective data show that Alemtuzumab based conditioning regimen in myeloid disorders have low incidence of GVHD, very low risk of graft rejection and comparable overall survival to those conditioning regimens which utilize in vivo T-cell depletion strategies other than Alemtuzumab. Moreover, a high incidence of viremia, in our cohort, did not translate into worse overall survival. DisclosuresSaif:Novartis: Honoraria; Alexion: Honoraria. Dignan:Jazz pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Therakos: Honoraria, Speakers Bureau. Tholouli:Jazz Pharmaceuticals: Honoraria, Speakers Bureau; MSD: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau.

A case of refractory rheumatoid pericarditis

A case of refractory rheumatoid pericarditis

Reply to V. Procházka et al

In their letter, Prochazka et al raise several issues as to the validity of our conclusion that in patients with relapsed or refractory follicular lymphoma, the presence of tumor cells in blood or bone marrow—as detected by BCL2/IgH major break point region polymerase chain reaction (PCR) at the end of rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone induction therapy—is not predictive of progression-free survival (PFS). First, they suggest that we should have analyzed separately patients with clinical complete (CR) and partial remission (PR). We agree that this would have been a relevant subgroup analysis. Unfortunately, the four subgroups (PCRpositive CR, PCR-negative CR, PCR-positive PR, and PCR-negative PR) were too small to allow for a meaningful statistical comparison. However, as mentioned in our report, there was no difference between patients in clinical CR or PR with regard to the proportion of bone marrow (BM) or peripheral blood PCR negativity at the end of induction treatment (50% v 55%). This is clearly indicative of the compartmentalization effect and at least partly explains the lack of prognostic value of PCR status post induction treatment. Second, the authors state that our finding that PFS was not different between patients who converted to PCR negativity and those who did not is in contrast with previous publications by others. We agree and consider this to be an important message of our report. Prochazka et al mention two observations in our article that they consider to be supportive of the predictive value of PCR status. Their first consideration is based on our finding that there was highly significant improved PFS by rituximab maintenance in patients with postinduction PCR-negative BM, whereas the improvement was not significant in patients with PCR-positive BM (Figs 4A, 4B). In fact, there was a clear difference in the PCR-positive BM group, but this did not reach statistical significance because of limited numbers. However, highly significant improvement in PFS by rituximab maintenance was observed in both patients with postinduction PCR-negative blood and those with postinduction PCR-positive blood (Figs 4C, 4D). These observations do not change our conclusion that benefit from rituximab maintenance is independent of postinduction PCR status. Second, they mention our observation that patients who were still PCR positive at the end of maintenance treatment progressed rapidly after stopping maintenance therapy. In our opinion, this observation is not indicative of the predictive value of postinduction PCR status, but it is a strong argument for prolonging maintenance treatment.

An International Survey of Cytomegalovirus Management Practices in Lung Transplantation

Cytomegalovirus (CMV) is an important infection in lung transplant recipients. Center-to-center variation in preventive and treatment strategies is unknown. An electronic survey was sent to 102 lung transplant programs registered with the International Society of Heart and Lung Transplantation and United Network for Organ Sharing. Fifty-nine (58%) programs responded to the survey. For CMV prevention (D+/R-), 56 of the 59 (94.9%) programs used prophylaxis and two (3.4%) of them used preemptive therapy. For R+ patients, 86.4% used prophylaxis and 13.6% used preemptive strategy. Duration of prophylaxis was extremely variable ranging from 3 months to indefinite. Adjunctive prophylactic strategies included routine viral monitoring (51% D+/R-; 44% R+) and CMV immunoglobulin (32% D+/R-; 14% R+). The medication used for prophylaxis was valganciclovir with approximately half starting with intravenous ganciclovir. 9 of the 59 (15.2%) centers reported using specific CMV prophylaxis in D-/R- patients. Methods for viral monitoring included peripheral blood polymerase chain reaction, antigenemia, bronchoalveolar lavage viral culture, and bronchoalveolar lavage polymerase chain reaction. For treatment of CMV viremia, valganciclovir or intravenous ganciclovir were used. A total of 47.5% of centers routinely decreased immunosuppression at the time of viremia. Secondary antiviral prophylaxis was used routinely by 36 of the 59 (61%) centers. Although prophylaxis is the most commonly used preventive strategy, significant variation exists in the way it is implemented. Specifically, duration of prophylaxis is extremely variable. Uniform international guidelines would be of value in this population.

Polymerase chain reaction of peripheral blood as a tool for the diagnosis of visceral leishmaniasis in children

The diagnosis of visceral leishmaniasis (VL) generally requires the use of invasive tests for the collection of infected tissue (aspirates of bone marrow, spleen, liver or lymph nodes). This difficulty has led to the search for safer and less painful techniques to confirm the occurrence of the disease in children. Polymerase chain reaction (PCR) is a method that is advantageous in that it allows the use of peripheral blood samples for diagnosis. This paper reports the utilisation of PCR on peripheral blood samples to diagnose VL in 45 children in Mato Grosso do Sul, Brazil. This technique is compared with methods carried out using tissue collected by invasive procedures, including direct microscopy, culture and detection of Leishmania DNA by PCR in bone marrow aspirates. The results show that PCR of peripheral blood provides great sensitivity (95.6%) that is similar to that from the PCR of bone marrow aspirates (91.1%) and higher than that achieved with microscopy (80%) or culture (26.7%) methods. PCR of peripheral blood proved to be a suitable tool for the diagnosis of VL in children because it is highly sensitive and safe, with tissue collection being less invasive than in traditional tests.

PARVOVIRUS B19-INDUCED PERSISTENT PURE RED CELL APLASIA IN A CHILD WITH T-CELL IMMUNODEFICIENCY

Persistent pure red cell aplasia can be a manifestation of parvovirus B19 infection in immunocompromised hosts. Failure of the humoral immune response to clear parvovirus B19 in such patients results in persistent pure red cell aplasia. The authors describe a child who had T-cell immunodeficiency and persistent pure red cell aplasia due to parvovirus B19 infection. Interestingly, they detected human parvovirus B19 genome by polymerase chain reaction (PCR) not in the peripheral blood, but in the bone marrow specimen of the patient. In their patient, T-cell immunodeficiency may have caused impaired B-cell activation and failure of effective humoral immune response to neutralize the virus. Additionally, before the diagnosis of pure red cell aplasia, IVIG treatment given at a dosage of 400 mg/kg/day with 3-week intervals may result in sufficient neutralization of peripheral blood parvovirus B19, whereas it may not be sufficient for the neutralization of parvovirus B19 genome in bone marrow. Thus, peripheral blood parvovirus B19 serology (IgM and IgG) and PCR were negative, whereas bone marrow aspiration sample was positive for parvovirus B19 PCR in this patient. Reticulocytopenia and severe anemia may warn the physicians of parvovirus B19 infection, especially in immunocompromised children. Diagnosis may require demonstration of absence of late erythroid precursors in the bone marrow as well as serologic testing and detection of parvovirus B19 genome by PCR in the serum and/or bone marrow samples of the patient.

Commercial LightCycler®-based quantitative real-time PCR compared to nested PCR for monitoring of Bcl-2/IgH rearrangement in patients with follicular lymphoma

Translocation of chromosomes 14 and 18 [t(14;18)] for detection of minimal residual disease in follicular lymphoma patients can be analyzed by nested polymerase chain reaction (PCR) or by quantitative PCR like LightCycler-based assays. We have compared both methods in blood and bone marrow samples of 28 patients enrolled in a clinical study on immunochemotherapy. In 42% of samples, the bcl2-IgH rearrangement was detectable by nested PCR, but not by LightCycler PCR. Nested PCR was able to reveal a significant drop in positive bone marrow or peripheral blood samples after therapy. In contrast, with LightCycler PCR, the detected drop in t(14;18)-positive cells did not reach statistical significance. The majority of patients showed positive results with nested PCR of peripheral blood or bone marrow without any associations to presence or absence of histological bone marrow (BM) infiltration by lymphoma cells. With LightCycler PCR, the numbers of positive cells were higher in samples from patients with BM infiltration of lymphoma cells (1.9 x 10(-2)) compared to samples from patients without involvement (4.08 x 10(-5)). A similar trend was seen in samples derived from the peripheral blood. Positivity for t(14;18) after therapy in two patients correlated with clinical relapse 6 months later. The data shown here demonstrate a lower sensitivity of LightCycler vs. nested PCR for detection of t(14;18). The usefulness of nested PCR for t(14;18) for risk stratification after primary therapy has to be validated in larger trials.

Evaluation of three polymerase chain reaction techniques for detection ofBrucellaDNA in peripheral human blood

Brucellosis is a widespread zoonosis. Currently the diagnosis of this zoonosis is based on microbiological and serological laboratory tests. Polymerase chain reaction (PCR) has been used to detect DNA from Brucella. Different target genes, primer pairs, PCR techniques, and extraction procedures have previously been published for Brucella detection. But only a few of these primers have been used in human samples, and only one study has been carried out to compare sensitivity between them. In the present study, 3 sets of primers and 3 different PCR protocols amplifying 3 different regions of the Brucella genome were compared for detection of Brucella DNA in a peripheral-blood PCR assay to conclude which is most suitable for the clinical diagnostic laboratory. These 3 pairs of primers amplify 3 different fragments included in (i) a gene encoding a 31 kDa Brucella abortus antigen (B4/B5), (ii) a sequence 16S rRNA of B. abortus (F4/R2), and (iii) a gene encoding an outer membrane protein (omp-2) (JPF/JPR). Some modifications on the reported techniques were applied during the present work to improve the outcome. The results showed that the B4/B5 primer pair had the highest sensitivity for detection of positive samples (98%), the JPF/JPR primer pair detected 88.4% of positive samples, whereas F4/R2 primer pair was the least sensitive, being able to detect only 53.1% of positive samples. The specificity of the 3 techniques was 100%. The B4/B5 primer pair was also able to detect the smallest number of bacteria (700 cfu/mL), whereas JPF/JPR was able to detect 7 x 105 cfu/mL and F4/R2 was able to detect 7 x 107 cfu/mL. It is thus concluded that using the B4/B5 primer PCR with the suggested modifications is a robust assay, which meets the sensitivity requirements to be used for testing of human blood samples for brucellosis in the diagnostic laboratory.

Relevance of Specific Immunoglobulin M and Immunoglobulin G for Parvovirus B19 Diagnosis in Patients With Acute Lymphoblastic Leukemia Receiving Chemotherapy: Prospective Study

Immunocompromised patients suffer from prolonged viral infections often without detectable immune response. However, even if the immune response occurs, can it clear the virus completely? To detect parvovirus B19 DNA and its antibodies in bone marrow cells and in serum by polymerase chain reaction (PCR) in children with acute lymphoblastic leukemia receiving chemotherapy to highlight the relation of humoral immune response to the presence of viremia. Also, to evaluate the optimal diagnostic test(s) for a correct diagnosis of parvovirus B19 disease in immunocompromised patients. Forty-eight children with acute lymphoblastic leukemia receiving maintenance chemotherapy were included in the study in addition to 20 healthy children with matched age and sex. Study for parvovirus B19 was performed by serologic determination of specific immunoglobulin (Ig) M and IgG, and viral DNA was determined by PCR in both serum and bone marrow aspiration. Parvovirus B19 DNA was detected in both serum and bone marrow in 20% of patients. Specific IgG was found in 40% and IgM in 26.7%. Two cases (10%) in the control group were positive for IgG. The agreement between IgG and positive results of PCR in the bone marrow was 33.3%, and the agreement for IgM and PCR in the serum was 33.3%. Parvovirus B19 is considered a common viral infection in children with acute lymphoblastic leukemia receiving chemotherapy. We must use our full potential to exclude such infection, which can mimic the side effects of chemotherapy in these patients. In immunocompromised patients, there are immunologic discrepancies in humoral immune responses for both IgM and IgG between individuals with parvovirus B19 persistence and healthy individuals, findings that may reflect both failed immunity and antigenic exhaustion. The contemporaneous determination of parvovirus B19 DNA by PCR in both bone marrow and peripheral blood and specific serologic markers appears to be the most appropriate diagnostic protocol for the correct laboratory diagnosis of parvovirus B19 infection in these patients.

Prognostic significance of circulating tumor cells in bone marrow or peripheral blood as detected by qualitative and quantitative PCR in pediatric NPM-ALK–positive anaplastic large-cell lymphoma

AbstractClinical and histopathological characteristics have limited prognostic value for children with anaplastic large-cell lymphoma (ALCL). We evaluated the presence, extent, and prognostic impact of circulating tumor cells in bone marrow (BM) and peripheral blood (PB) of children and adolescents with NPM-ALK–positive ALCL at diagnosis using qualitative and quantitative polymerase chain reaction (PCR) for NPM-ALK. Numbers of NPM-ALK transcripts were normalized to 104 copies ABL (NCNs). BM was analyzed from 80 patients and PB from 52. BM was positive for NPM-ALK in 47.5% of patients, and positivity was significantly correlated with clinical stage, mediastinal or visceral involvement, microscopic BM involvement, and histologic subtype. Qualitative and quantitative PCR results in BM and PB strongly correlated. BM PCR was associated with the cumulative incidence of relapses (CI-Rs): CI-R was 50% ± 10% for 38 PCR-positive and 15% ± 7% for 42 PCR-negative patients (P < .001). Sixteen patients with more than 10 NCNs NPM-ALK in BM had a CI-R of 71% ± 14% compared with a CI-R of 18% ± 6% for 59 patients with 10 or fewer NCNs (P < .001). PB PCR results led to a similar grouping. Thus, quantitative PCR in BM or PB allows identification of 20% of patients experiencing 60% of all relapses with an event-free survival of 20%.

Using polymerase chain reaction in early diagnosis of re-activated Trypanosoma cruzi infection after heart transplantation

Background Heart transplantation is an effective treatment for patients with end-stage Chagas’ heart disease. Re-activation of Chagas’ disease in transplant recipients is frequent, triggered by immunosuppression level. Therefore, highly sensitive methods for early diagnosis of Chagas’ disease relapse are necessary to initiate appropriate therapy. We analyzed the use of polymerase chain reaction (PCR) in the clinical follow-up of heart transplant recipients. Methods We prospectively evaluated 4 heart transplant recipients at the Hospital Privado, Córdoba, Argentina, who had terminal Chagas’ disease. The parameters analyzed were presence of parasites in the blood (blood culture, Strout) and in endomyocardial biopsy (EMB) samples, and PCR was performed with oligonucleotides directed to a nuclear repetitive sequence of Trypanosoma cruzi. We evaluated these parameters weekly from the day of transplantation until results were negative and then during regular follow-up visits. Results In 2 patients, we detected T cruzi using PCR in peripheral blood 30 days before clinical evidence of re-activation. In the 3rd case, PCR results in peripheral blood were positive from the day before transplantation, followed by positive results in EMB and sub-cutaneous chagomas biopsy specimens. Only 1 patient had positive Strout results for parasites in skin lesions, and none showed amastigotes in the biopsy specimens. After clinical diagnosis, all patients received 5 mg/kg/day benzimidazole for 6 months, with acceptable tolerance and good clinical outcome. All patients had negative peripheral blood PRC results after 30 days of treatment. One patient had intermittent positive PCR results during follow-up, with no evidence of clinical re-activation. Conclusion Polymerase chain reaction detection of T Cruzi in heart transplant recipients is a more sensitive and specific procedure in diagnosing Chagas’ disease re-activation.

Fludarabine, Cyclophosphamide and Mitoxantrone for Untreated Follicular Lymphoma: a Report from the Non-Hodgkin's Lymphoma Co-operative Study Group

The aim of the study was to determine the safety and efficacy of the combination of fludarabine (FLU), cyclophosphamide (CY) and mitoxantrone (FLU/CY/MITO) in untreated follicular lymphomas (FL), Sixty patients with newly diagnosed stage II bulky to IV FL, median age 59 years (range 36-70), received FLU/CY/MITO regimen (FLU 25 mg/m2 days 1-3, CY 300 mg/m2 days 1-3, Mito 10 mg/m2 day 1). Patients received antibiotic oral prophylaxis during all treatments, and growth factors (G-CSF) when grade III granulocytopenia (WHO) occurred. The overall response rate was 87%: 46 patients achieved complete response (CR) (77%), 6 a partial response (10%) and 8 were non-responders. Fifty patients are surviving with a median observation time of 31 months. The 4-year estimated probability of overall survival and failure-free survival were 78.2% and 45% respectively. Thirty-five patients (58%) are still in CR. Sixty percent of patients experienced grade III-IV granulocytopenia. Two patients suffered grade III pulmonary infection and one grade III liver toxicity. In a subset of 46 patients, bcl-2 translocation was positive in bone marrow (BM) and/or peripheral blood (PB) of 36 patients. At the end of treatment, 25 of these patients had CR and 19 (76%) converted to polymerase chain reaction (PCR) negativity. FLU/CY/MITO regimen showed a high level of activity in follicular lymphoma. Toxicity, mainly hematological, was acceptable and the treatment was made feasible by the use of antibiotic prophylaxis and G-CSF. Significant non-hematological toxicities were seen, but no patients died. The conversion of bcl-2 from positive to negative by PCR in BM and/or PB suggests a possible role for this treatment in clearing minimal residual disease and improving patients' outcome.

Epstein-Barr virus and post-transplant lymphoproliferative disease.

There is convincing evidence that Epstein-Barr virus (EBV) is associated with post-transplant lymphoproliferative disease (PTLD). Primary EBV infection following transplantation occurs in as many as 90% of cases of PTLD in children and pretransplant EBV seronegativity is a recognized risk factor for developing PTLD. Other risk factors include young age at the time of transplant, the type of transplant that the recipient receives and the type and intensity of immunosuppression. The clinical presentation is often nonspecific and tissue biopsy is necessary to establish the diagnosis. There appears to be a correlation between PTLD and EBV viral load measured by polymerase chain reaction (PCR) of the peripheral blood and quantitative PCR may be a useful guide in the management of PTLD. Antiviral drugs and cytomegalovirus-immunoglobulin G may have a role in preventing PTLD. Because PTLD results from functional over-immunosuppression, the initial treatment is reduction of immunosuppression. Antiviral agents, interferon, immuno-based monoclonal therapy, cell-based therapy and chemotherapy also have a potential role in treating this disorder. At the present time there is no standardized approach to the evaluation and treatment of PTLD.