Evaluation and additional recommendations for preparing a whole blood control material

OBJECTIVE: The assessment of an easy to prepare and low cost control material for Hematology, available for manual and automated methods. MATERIAL AND METHOD: Aliquots of stabilized whole blood were prepared by partial fixation with aldehydes; the stability at different temperatures (4. 20 and 37 °C) during periods of up to 8-9 weeks and aliquot variability with both methods were controlled. RESULTS: Aliquot variability with automated methods at day 1, expressed as CV% (coefficient of variation) was: white blood cells (WBC) 2.7, red blood cells (RBC) 0.7, hemoglobin (Hb) 0.6, hematocrit (Hct) 0.7, mean cell volume (MCV) 0.3, mean cell hemoglobin (MCH) 0.6, mean cell hemoglobin concentration (MCHC) 0.7, and platelets (PLT) 4.6. The CV (coefficient of variation) percentages obtained with manual methods in one of the batches were: WBC 23, Hct 2.8, Hb 4.5, MCHC 5.9, PLT 41. Samples stored at 4ºC and 20ºC showed good stability, only a very low initial hemolysis being observed, whereas those stored at 37ºC deteriobed a rapidly (metahemoglobin formation, aggregation of WBC and platelets, as well as alteration of erythrocyte indexes). CONCLUSIONS: It was confirmed that, as long as there is no exposure to high temperatures during distribution, this material is stable, allowing assessment, both esternal and internal, for control purposes, with acceptable reproductivity, both for manual and auttomatic methods.

Cell mediated immune response in human antirabies revaccination

The occurrence of secondary cell mediated immune response (CMI) in human antirabies immunization was studied. The Puenzalida & Palácios vaccine was used because it is routinely used in Brazil. CMI was evaluated by lymphoblastic transformation indices obtained in whole blood culture in the presence of rabies and control (nervous tissue) antigens. Eleven volunteers submitted to revaccination constituted the group under study, while three other volunteers submitted primo vaccination were utilized as control group. A clear secondary CMI to rabies antigen was detected in all the revaccinated volunteers who showed earlier and more intense response than the control group. Response to the control antigen, however, present in all the components of the first group was not detectable in two out of the three primovaccinated and very low in the third one.

Cell and humoral immunity in endemic pemphigus foliaceus

A study was conducted on 16 patients with pemphigus foliaceus, ten of them with the localized form (group G1) and six with the disseminated form (group G2). These patients were submitted to full blood counts, quantitation of mononuclear cell subpopulations by monoclonal antibodies, study of blastic lymphocyte transformation, and quantitation of circulating antibodies by the indirect immunofluorescence test, in order to correlate their clinical signs and symptoms and laboratory data with their immunological profile, and to determine the relationship between circulating autoantibody titers and lesion intensity and course of lesions under treatment. Leucocytosis was observed especially in group G2. All patients showed decreased relative CD3+ and CD4+ values and a tendency to decreased relative values of the CD8+ subpopulation. Blastic lymphocyte transformation indices in the presence of phytohemagglutinin were higher in patients (group G1+G2) than in controls. The indirect immunofluorescence test was positive in 100% of G2 patients and in 80% of G1 patients. The median value for the titers was higher in group G2 than in group G1. Analysis of the results as a whole permits us to conclude that cell immunity was preserved and that there was a relationship between antibody titers detected by the direct immunofluorescence test and extent of skin lesions.

Assessment of PCR in the detection of Leishmania spp in experimentally infected individual phlebotomine sandflies (Diptera: Psychodidae: Phlebotominae)

DNA amplification by the polymerase chain reaction (PCR) was applied in the investigation of the presence of Leishmania (Kinetoplastida: Trypanosomatidae) parasites in single phlebotomine sandflies. Three phlebotomine/parasite pairs were used: Lutzomyia longipalpis/Leishmania chagasi, Lutzomyia migonei/Leishmania amazonensis and Lutzomyia migonei/Leishmania braziliensis, all of them incriminated in the transmission of visceral or cutaneous leishmaniasis. DNA extraction was performed with whole insects, with no need of previous digestive tract dissection or pooling specimens. The presence of either mouse blood in the digestive tract of the sandflies or the digestive tract itself did not interfere in the PCR. Infection by as few as 10 Leishmania sp. per individual were sufficient for DNA amplification with genus-specific primers. Using primers for L. braziliensis and L. mexicana complexes, respectively, it was possible to discriminate between L. braziliensis and L. amazonensis in experimentally infected vectors (L. migonei).

Neurocryptococcosis: diagnosis by PCR method

Cryptococcus neoformans detection was optimized using PCR technique with the objective of application in the clinical laboratory diagnosis. The amplification area was ITS and 5,6S which encodes the ribosomal RNA (rRNA). A total of 72 cerebrospinal fluid (CSF) samples were used, obtained from cases with and without AIDS. The patients had cryptococcal meningitis (n = 56) and meningitis caused by other agents (n = 16). The results demonstrated that PCR test had the highest sensitivity rates, superior to culture (85.7%) and to India ink test (76.8%). PCR was found to be sensitive in detecting 1 cell/mL and highly specific since it did not amplify other fungal DNA. The comparative analysis of the methods showed that PCR is more sensitive and specific and is applicable as an important laboratorial resource for neurocryptococcosis diagnosis.

Detection of EBV-DNA in serum samples of an immunosuppressed child during a three years follow-up: association of clinical and PCR data with active infection

Twenty-four whole blood and serum samples were drawn from an eight year-old heart transplant child during a 36 months follow-up. EBV serology was positive for VCA-IgM and IgG, and negative for EBNA-IgG at the age of five years old when the child presented with signs and symptoms suggestive of acute infectious mononucleosis. After 14 months, serological parameters were: positive VCA-IgG, EBNA-IgG and negative VCA-IgM. This serological pattern has been maintained since then even during episodes suggestive of EBV reactivation. PCR amplified a specific DNA fragment from the EBV gp220 (detection limit of 100 viral copies). All twenty-four whole blood samples yielded positive results by PCR, while 12 out of 24 serum samples were positive. We aimed at analyzing whether detection of EBV-DNA in serum samples by PCR was associated with overt disease as stated by the need of antiviral treatment and hospitalization. Statistical analysis showed agreement between the two parameters evidenced by the Kappa test (value 0.750; p < 0.001). We concluded that detection of EBV-DNA in serum samples of immunosuppressed patients might be used as a laboratory marker of active EBV disease when a Real-Time PCR or another quantitative method is not available.

Reproduction of Leishmania (Leishmania ) infantum chagasi in conditioned cell culture growth medium

Leishmanias can be produced by inoculation in conditioned McCoy cell culture growth medium (CGM). Leishmania (Leishmania) infantum chagasi (100 parasites) grown in NNN medium was inoculated in 2.5 mL CGM, kept in plates (24 wells) and its multiplication was observed for five days (120 hours). After day 5, the medium was saturated with the flagellate forms of the parasite (promastigotes). The reproduction of the leishmanias was observed every 24 hours and the number of parasites was calculated by counting the parasites in a drop of 10 µ L and photomicrographied. So the number of Leishmanias was adjusted to 1 mL volume. The advantage of the technique by isolation of Leishmania in CGM demonstrated in this study is its low cost and high efficacy even with a small quantity of parasites (10² promastigotes) used as inoculum. Additionally, isolation of the leishmania can be obtained together with an increase in their density (180 times) as observed by growth kinetics, within a shorter time. These results justify the use of this low-cost technique for the isolation and investigation of the behavior and multiplication of Leishmania both in vertebrates and invertebrates, besides offering means of obtaining antigens, whether whole antigens (leishmanias) or the soluble antigens produced by the parasites which may be useful for the production of new diagnostic kits.

Detection of HIV and HCV RNA in semen from Brazilian coinfected men using multiplex PCR before and after semen washing

INTRODUCTION: Prolonged survival of patients under HAART has resulted in new demands for assisted reproductive technologies. HIV serodiscordant couples wish to make use of assisted reproduction techniques in order to avoid viral transmission to the partner or to the newborn. It is therefore essential to test the effectiveness of techniques aimed at reducing HIV and HCV loads in infected semen using molecular biology tests. METHODS: After seminal analysis, semen samples from 20 coinfected patients were submitted to cell fractioning and isolation of motile spermatozoa by density gradient centrifugation and swim-up. HIV and HCV RNA detection tests were performed with RNA obtained from sperm, seminal plasma and total semen. RESULTS: In pre-washing semen, HIV RNA was detected in 100% of total semen samples, whereas HCV RNA was concomitantly amplified in only one specimen. Neither HIV nor HCV were detected either in the swim-up or in the post-washing semen fractions. CONCLUSIONS: Reduction of HIV and/or HCV shedding in semen by density gradient centrifugation followed by swim-up is an efficient method. These findings lead us to believe that, although semen is rarely found to contain HCV, semen processing is highly beneficial for HIV/HCV coinfected individuals.

Comparison of serology, antigenemia assay and the polymerase chain reaction for monitoring active cytomegalovirus infections in hematopoietic stem cell transplantation patients

Forty-six allogeneic hematopoietic stem cell transplantation (HSCT) patients were monitored for the presence of CMV antibodies, CMV-DNA and CMV antigens after transplantation. Immunoenzymatic serological tests were used to detect IgM and the increase in CMV IgG antibodies (increase IgG), a nested polymerase chain reaction (N-PCR) was used to detect CMV-DNA, and an antigenemia assay (AGM) was used to detect CMV antigens. The presence of CMV-IgM and/or CMV-increase IgG antibodies was detected in 12/46 (26.1%) patients, with a median time between HSCT and the detection of positive serology of 81.5 days. A positive AGM was detected in 24/46 (52.2%) patients, with a median time between HSCT and antigen detection of 62 days. Two or more consecutive positive N-PCR results were detected in 32/46 (69.5%) patients, with a median time between HSCT and the first positive PCR of 50.5 days. These results confirmed that AGM and mainly PCR are superior to serology for the early diagnosis of CMV infection. Six patients had CMV-IgM and/or CMV-increase IgG with a negative AGM (five cases) or N-PCR assay (one case). In five of these cases the serological markers were detected during the first 100 days after HSCT, the period of highest risk. These findings support the idea that serology may be useful for monitoring CMV infections in HSCT patients, especially when PCR is unavailable.

Kinetics of growth of Leishmania (Leishmania) chagasi cycle in McCoy cell culture

The kinetics of growth of Leishmania performed in vitro after internalization of the promastigote form in the cell and the occurrence of the transformation of the parasite into the amastigote form have been described by several authors. They used explants of macrophages in hamster spleen cell culture or in a human macrophage lineage cell, the U937. Using microscopy, the description of morphologic inter-relationship and the analysis of the production of specific molecules, it has been possible to define some of the peculiarities of the biology of the parasite. The present study shows the growth cycle of Leishmania chagasi during the observation of kinetic analysis undertaken with a McCoy cell lineage that lasted for a period of 144 hours. During the process, the morphologic transformation was revealed by indirect immunofluorescence (IF) and the molecules liberated in the extra cellular medium were observed by SDS-PAGE at 24-hour intervals during the whole 144-hour period. It was observed that in the first 72 hours the promastigote form of L. chagasi adhered to the cell membranes and assumed a rounded (amastigote-like) form. At 96 hours the infected cells showed morphologic alterations; at 120 hours the cells had liberated soluble fluorescent antigens into the extra cellular medium. At 144 hours, new elongated forms of the parasites, similar to promastigotes, were observed. In the SDS-PAGE, specific molecular weight proteins were observed at each point of the kinetic analysis showing that the McCoy cell imitates the macrophage and may be considered a useful model for the study of the infection of the Leishmania/cell binomial.

T CD4+ cells count among patients co-infected with human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1): high prevalence of tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM)

INTRODUCTION: HIV positive patients co-infected with HTLV-1 may have an increase in their T CD4+ cell counts, thus rendering this parameter useless as an AIDS-defining event. OBJECTIVE: To study the effects induced by the co-infection of HIV-1 and HTLV-1 upon CD4+ cells. MATERIAL AND METHODS: Since 1997, our group has been following a cohort of HTLV-1-infected patients, in order to study the interaction of HTLV-1 with HIV and/or with hepatitis C virus (HCV), as well as HTLV-1-only infected asymptomatic carriers and those with tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM). One hundred and fifty HTLV-1-infected subjects have been referred to our clinic at the Institute of Infectious Diseases "Emílio Ribas", São Paulo. Twenty-seven of them were also infected with HIV-1 and HTLV-1-infection using two ELISAs and confirmed and typed by Western Blot (WB) or polymerase chain reaction (PCR). All subjects were evaluated by two neurologists, blinded to the patient's HTLV status, and the TSP/HAM diagnostic was based on the World Health Organization (WHO) classification. AIDS-defining events were in accordance with the Centers for Disease Control (CDC) classification of 1988. The first T CD4+ cells count available before starting anti-retroviral therapy are shown compared to the HIV-1-infected subjects at the moment of AIDS defining event. RESULTS: A total of 27 HIV-1/HTLV-1 co-infected subjects were identified in this cohort; 15 already had AIDS and 12 remained free of AIDS. The median of T CD4+ cell counts was 189 (98-688) cells/mm³ and 89 (53-196) cells/mm³ for co-infected subjects who had an AIDS-defining event, and HIV-only infected individuals, respectively (p = 0.036). Eight of 27 co-infected subjects (30%) were diagnosed as having a TSP/HAM simile diagnosis, and three of them had opportunistic infections but high T CD4+ cell counts at the time of their AIDS- defining event. DISCUSSION: Our results indicate that higher T CD4+ cells count among HIV-1/HTLV-1-coinfected subjects was found in 12% of the patients who presented an AIDS-defining event. These subjects also showed a TSP/HAM simile picture when it was the first manifestation of disease; this incidence is 20 times higher than that for HTLV-1-only infected subjects in endemic areas.

Comparison of direct immunofluorescence, conventional cell culture and polymerase chain reaction techniques for detecting respiratory syncytial virus in nasopharyngeal aspirates from infants

A total of 316 samples of nasopharyngeal aspirate from infants up to two years of age with acute respiratory-tract illnesses were processed for detection of respiratory syncytial virus (RSV) using three different techniques: viral isolation, direct immunofluorescence, and PCR. Of the samples, 36 (11.4%) were positive for RSV, considering the three techniques. PCR was the most sensitive technique, providing positive findings in 35/316 (11.1%) of the samples, followed by direct immunofluorescence (25/316, 7.9%) and viral isolation (20/315, 6.3%) (p < 0.001). A sample was positive by immunofluorescence and negative by PCR, and 11 (31.4%) were positive only by RT-PCR. We conclude that RT-PCR is more sensitive than IF and viral isolation to detect RSV in nasopharyngeal aspirate specimens in newborn and infants.

The monitoring of hematopoietic stem cell transplant donors and recipients from endemic areas for malaria

Malaria is an unusual complication after hematopoietic stem cell transplantation in non-endemic countries. However, transplant candidates, recipients and donors living in endemic regions frequently report previous episodes of malaria. This fact could represent an important risk for immunosuppressed recipients that could develop severe malaria cases. We report a case of hematopoietic stem cell transplant (HSCT) in which the donor had a history of previous malaria, and close monitoring was performed before and after procedure by parasitological and molecular tests. The donor presented Plasmodium vivax in thick blood smears one month after transplant and was treated according to Brazilian Health Ministry guidelines. The polymerase chain reaction (PCR) was able to detect malaria infection in the donor one week earlier than thick blood film. Even without positive results, the recipient was pre-emptively treated with chloroquine in order to prevent the disease. We highlight the importance of monitoring recipients and donors in transplant procedures with the aim of reducing the risk of malaria transmission.

Adenovirus respiratory infection: significant increase in diagnosis using PCR comparing with antigen detection and culture methods

Adenovirus (AdV) respiratory infections are usually described as being associated with high mortality rates. Laboratory diagnosis is essential for the establishment of the appropriate therapy, and for guiding the implementation of preventive measures in order to prevent the spread of the infection. Aiming to analyze the sensitivity and specificity of the laboratorial diagnosis methods available, we compared antigen detection by indirect immunofluorescence assay (IF), and a specific nested polymerase chain reaction (PCR), to detect AdV in respiratory samples collected from patients admitted to hospital with acute respiratory disease. Positive samples were inoculated into a cell culture to confirm the results. We analyzed 381 samples from the nasopharyngeal aspirates collected during the year 2008; of these, 2.6% tested were positive for adenovirus through IF and 10% through PCR; positive isolation was obtained in 40% and 26% of these cases, respectively. Most infected patients were children under six months of age, and despite of the fact that a significant number of patients required intensive care, the mortality rate was low (5%). In conclusion, molecular methods were found to be useful for rapid diagnosis of adenovirus infections with higher sensitivity than antigen detection; their introduction permitted a significant increase in diagnoses of adenovirus infections.

The value of in vitro cell culture of granulocytes in the detection of Ehrlichia

Peripheral blood leukocytes from different animals were isolated from whole blood and maintained in Dulbeco's medium containing homologous serum without antibiotics. After 72 hrs microscopic examination of these cells showed that most animals were infected with Ehrlichia. Observation of thin blood smears from the same animals showed that only two were positive for Ehrlichia. The results of this investigation show that leukocyte culture is superior to the traditional thin blood film method in the detection of Ehrlichia and that asymptomatic carriers are easily detected. The method is inexpensive and does not require specific cell lines although it is necessary to use sterile sera.