Antiretroviral resistance among HIV type 1-infected women first exposed to antiretrovirals during pregnancy: Plasma versus PBMCs

Resistance-associated mutations (RAMs) in plasma samples from HIV-1-infected women who received antiretroviral (ARV) prophylaxis during pregnancy was assessed and correlated with the detection of RAMs in peripheral blood mononuclear cells (PMBCs). The study population was composed of HIV-1-infected women enrolled in a prospective cohort study in Latin America and the Caribbean (NISDI Perinatal Study) as of March 1, 2005, who were diagnosed with HIV-1 infection during the current pregnancy, who received ARVs during pregnancy for prevention of mother-to-child transmission of HIV-1, and who were followed through at least the 6-12 week postpartum visit. Plasma samples collected at enrollment during pregnancy and at 6-12 weeks postpartum were assayed for RAMs. Plasma results were compared to previously described PBMC results from the same study population. Of 819 enrolled subjects, 197 met the eligibility criteria. Nucleic acid amplification was accomplished in 123 plasma samples at enrollment or 6-12 weeks postpartum, and RAMs were detected in 22 (17.9%; 95% CI: 11.7-25.9%). Previous analyses had demonstrated detection of RAMs in PBMCs in 19 (16.1%). There was high concordance between RAMs detected in plasma and PBMC samples, with only eight discordant pairs. The prevalence of RAMs among these pregnant, HIV-1-infected women is high (>15%). Rates of detection of RAMs in plasma and PBMC samples were similar.

Immunohistochemistry and polymerase chain reaction on paraffin-embedded material improve the diagnosis of cutaneous leishmaniasis in the Amazon region

Background Recently, there has been an increase in the incidence of cutaneous leishmaniasis (CL), which represents an important health problem. This increase may be related to the epidemiologic expansion of the infective agent and the increase in tourism in tropical areas. The difficulty in clinical diagnosis, mainly in areas in which CL is not the first consideration of local physicians, has intensified efforts to describe diagnostic tests, which should be specific, sensitive, and practical. Amongst the new tests described are those including nucleic acid amplification (polymerase chain reaction, PCR) and immunohistochemistry (IHC). Methods In this study, we evaluated the sensitivity of a PCR based on small subunit (SSU) ribosomal DNA, in comparison with IHC using Leishmania spp. antibodies, in biopsies embedded in paraffin. Result The results indicated a total sensitivity of 96% (90.9% with PCR and 68.8% with IHC), showing the possibility of using paraffin-embedded biopsies to diagnose CL. Conclusion We propose the use of the two tests together as a routine protocol for diagnosis. This would require the provision of local medical services to perform molecular biology techniques and adequate Leishmania antibodies.

Modern scientific methods and their potential in wastewater science and technology

Application of novel analytical and investigative methods such as fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM), microelectrodes and advanced numerical simulation has led to new insights into micro-and macroscopic processes in bioreactors. However, the question is still open whether or not these new findings and the subsequent gain of knowledge are of significant practical relevance and if so, where and how. To find suitable answers it is necessary for engineers to know what can be expected by applying these modern analytical tools. Similarly, scientists could benefit significantly from an intensive dialogue with engineers in order to find out about practical problems and conditions existing in wastewater treatment systems. In this paper, an attempt is made to help bridge the gap between science and engineering in biological wastewater treatment. We provide an overview of recently developed methods in microbiology and in mathematical modeling and numerical simulation. A questionnaire is presented which may help generate a platform from which further technical and scientific developments can be accomplished. Both the paper and the questionnaire are aimed at encouraging scientists and engineers to enter into an intensive, mutually beneficial dialogue. (C) 2002 Elsevier Science Ltd. All rights reserved.

Rapid diagnosis in pediatric infectious diseases: The past, the present and the future

The focus of rapid diagnosis of infectious diseases of children in the last decade has shifted from variations of the conventional laboratory techniques of antigen detection, microscopy and culture to that of molecular diagnosis of infectious agents. Pediatricians will need to be able to interpret the use, limitations and results of molecular diagnostic techniques as they are increasingly integrated into routine clinical microbiology laboratory protocols. PCR is the best known and most successfully implemented diagnostic molecular technology to date. It can detect specific infectious agents and determine their virulence and antimicrobial genotypes with greater speed, sensitivity and specificity than conventional microbiology methods. Inherent technical limitations of PCR are present, although they are reduced in laboratories that follow suitable validation and quality control procedures. Variations of PCR together with advances in nucleic acid amplification technology have broadened its diagnostic capabilities in clinical infectious disease to now rival and even surpass traditional methods in some situations. Automation of all components of PCR is now possible. The completion of the genome sequencing projects for significant microbial pathogens, in combination with PCR and DNA chip technology, will revolutionize the diagnosis and management of infectious diseases.

Light controlled synthesis of nucleic acids

Dissertação apresentada para obtenção do grau de Doutor em Bioquímica - especialidade Biotecnologia, pela Universidade Nova de Lisboa,Faculdade de Ciências e Tecnologia

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.

Replacement of HIV p24 Ag test by a multiplex RT-PCR method for primary screening of blood donors

Nucleic Acid Testing (NAT) as a tool for primary screening of blood donors became a reality in the end of the 1990 decade. We report here the development of an "in-house" RT-PCR method that allows the simultaneous (multiplex) detection of HCV and HIV-RNA in addition to an artificial RNA employed as an external control. This method detects all HIV group M subtypes, plus group N and O, with a detection threshold of 500 IU/mL. After validation, the method replaced p24 Ag testing, in use for blood donation screening since 1996 at our services. From July 2001 to February 2006, 102,469 donations were tested and 41 (0.04%) were found HIV-RNA reactive. One NAT-only reactive donation (antibody non-reactive) was observed, with subsequent seroconversion of the implied donor, giving a yield of 1:102,469. This rate is in contrast to the international experience that reports a detection of approximately 1:600,000 - 1:3,100,000 of isolated HIV-RNA donations.

Primary screening of blood donors by nat testing for HCV-RNA: development of an "in-house" method and results

An "in-house" RT-PCR method was developed that allows the simultaneous detection of the RNA of the Hepatitis C Virus (HCV) and an artificial RNA employed as an external control. Samples were analyzed in pools of 6-12 donations, each donation included in two pools, one horizontal and one vertical, permitting the immediate identification of a reactive donation, obviating the need for pool dismembering. The whole process took 6-8 hours per day and results were issued in parallel to serology. The method was shown to detect all six HCV genotypes and a sensitivity of 500 IU/mL was achieved (95% hit rate). Until July 2005, 139,678 donations were tested and 315 (0.23%) were found reactive for HCV-RNA. Except for five false-positives, all 310 presented the corresponding antibody as well, so the yield of NAT-only donations was zero, presenting a specificity of 99.83%. Detection of a window period donation, in the population studied, will probably demand testing of a larger number of donations. International experience is showing a rate of 1:200,000 - 1:500,000 of isolated HCV-RNA reactive donations.

MALARIA DIAGNOSIS BY LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) IN THAILAND

The loop-mediated isothermal amplification method (LAMP) is a recently developed molecular technique that amplifies nucleic acid under isothermal conditions. For malaria diagnosis, 150 blood samples from consecutive febrile malaria patients, and healthy subjects were screened in Thailand. Each sample was diagnosed by LAMP, microscopy and nested polymerase chain reaction (nPCR), using nPCR as the gold standard. Malaria LAMP was performed using Plasmodiumgenus and Plasmodium falciparum specific assays in parallel. For the genus Plasmodium, microscopy showed a sensitivity and specificity of 100%, while LAMP presented 99% of sensitivity and 93% of specificity. For P. falciparum, microscopy had a sensitivity of 95%, and LAMP of 90%, regarding the specificity; and microscopy presented 93% and LAMP 97% of specificity. The results of the genus-specific LAMP technique were highly consistent with those of nPCR and the sensitivity of P. falciparum detection was only marginally lower.

Métodos Preditivos de Metastização Ganglionar Adicional em Cancro da Mama com Gânglio Sentinela Positivo

Objectivos As recomendações para a realização de linfadenectomia axilar nos doentes com cancro da mama em estádio precoce e sujeitas a biópsia de gânglio sentinela com presença de macrometástase foram recentemente actualizadas, baseadas nos últimos estudos publicados (Z0011 e IBCSG 23-01). Mantém-se, no entanto, alguma controvérsia na decisão de não realização de cirurgia radical axilar nos doentes com gânglio sentinela positivo. Têm sido desenvolvidos métodos preditivos de metastização ganglionar adicional. Um dos mais conhecidos, desenvolvido no Memorial Sloan-Kettering Cancer Center (MSKCC), recorre a variáveis do tumor e características do gânglio sentinela. Mais recentemente com a utilização de métodos moleculares (como o One Step Nucleic Acid - OSNA) para estudo do gânglio sentinela tem sido avaliada a capacidade preditiva da quantidade total de cópias mRNA da citoqueratina 19. Pretende-se estudar na nossa amostra a capacidade preditiva do nomograma do MSKCC e da carga tumoral total. Propõe-se ainda avaliar o número de macrometástases encontradas na biópsia de gânglio sentinela e sua relação na metastização ganglionar adicional. Material e métodos Avaliação retrospectiva de 819 doentes com cancro da mama (Tis – T2) submetidos a biópsia de gânglio sentinela no Centro Hospitalar de Lisboa Central durante o período de 1 de Janeiro de 2005 e 31 de Dezembro de 2013. Foram identificados 123 doentes com gânglio sentinela positivo que realizaram linfadenectomia axilar imediata. A análise do gânglio sentinela foi executada por métodos histológicos em 78 doentes e por método molecular (OSNA) em 45 doentes. Os dois grupos foram estudados separadamente, tendo sido no primeiro aplicado o nomograma do MSKCC e no segundo obtida a carga tumoral total. Utilizou-se o modelo de regressão logística para analisar o poder preditivo e discriminativo destes dois métodos. Adicionalmente, para avaliar a potencial importância do número de macrometástases na metastização ganglionar adicional, foi ajustado um novo modelo de regressão logística considerando esta variável e a carga tumoral total. Ambos os métodos foram também avaliados através da área sob a curva ROC (AUC) e do teste de Hosmer-Lemeshow, respectivamente. O nível de significância adoptado foi α = 0.05. O estudo estatístico foi realizado com recurso ao SPSS. Resultados No grupo em que foi aplicado o nomograma do MSKCC obteve-se uma AUC=0.67 (I.C. 95% = 0.55 – 0.79), e no grupo em que foi avaliada a carga tumoral total uma AUC=0.78 (I.C. 95% = 0.64 – 0.91). Os poderes preditivos de ambos foram, respectivamente, p=0.15 e p=0.46. Constatou-se que o desempenho do modelo resultante da junção da carga tumoral total com o número de macrometástases encontradas no estudo do gânglio sentinela foi bastante satisfatório (AUC=0.87, I.C 95% = 0.76 – 0.98, poder preditivo p=0.33). Conclusão Foi validado externamente o modelo do MSKCC para a amostra em estudo, apresentando uma menor acuidade discriminativa em relação ao estudo original (AUC=0.67 versus AUC=0.75). Por outro lado, após verificação da homogeneidade de ambos os grupos no que diz respeito a todas as variáveis de interesse, conclui-se que a carga tumoral total aparenta uma maior acuidade preditiva e discriminativa na metastização ganglionar adicional que o nomograma do MSKCC. Quando desenvolvido um modelo agregando a carga tumoral total avaliada por OSNA e o número de macrometástases no gânglio sentinela, obteve-se uma capacidade discriminativa ainda superior. A carga tumoral total avaliada por OSNA, ou esta incluída num modelo conjunto com o número de macrometástases obtidas no estudo do gânglio sentinela, poderão representar importantes ferramentas preditivas. Serão no entanto necessários estudos adicionais com amostras superiores para consolidar estes resultados. Bibliografia 1. Giuliano AE, McCall L, Beitsch P, Whitworth PW, Blumencranz P, Leitch AM, et al. Locoregional recurrence after sentinel lymph node dissection with or without axillary dissection in patients with sentinel lymph node metastases: the American College of Surgeons Oncology Group Z0011 randomized trial. Ann Surg. Setembro de 2010;252(3):426–32; discussion 432–3. 2. Galimberti V, Cole BF, Zurrida S, Viale G, Luini A, Veronesi P, et al. Axillary dissection versus no axillary dissection in patients with sentinel-node micrometastases (IBCSG 23-01): a phase 3 randomised controlled trial. Lancet Oncol. Abril de 2013;14(4):297–305. 3. Bevilacqua JLB, Kattan MW, Fey JV, Cody HS, Borgen PI, Van Zee KJ. Doctor, what are my chances of having a positive sentinel node? A validated nomogram for risk estimation. J Clin Oncol. 20 de Agosto de 2007;25(24):3670–9. 4. Piñero A, Canteras M, Moreno A, Vicente F, Giménez J, Tocino A, et al. Multicenter validation of two nomograms to predict non-sentinel node involvement in breast cancer. Clin Transl Oncol. Fevereiro de 2013;15(2):117–23. 5. Banerjee SM, Michalopoulos NV, Williams NR, Davidson T, El Sheikh S, McDermott N, et al. Detailed evaluation of one step nucleic acid (OSNA) molecular assay for intra-operative diagnosis of sentinel lymph node metastasis and prediction of non-sentinel nodal involvement: experience from a London teaching hospital. Breast. Agosto de 2014;23(4):378–84. 6. Peg V, Espinosa-Bravo M, Vieites B, Vilardell F, Antúnez JR, de Salas MS, et al. Intraoperative molecular analysis of total tumor load in sentinel lymph node: a new predictor of axillary status in early breast cancer patients. Breast Cancer Res Treat. Maio de 2013;139(1):87–93. 7. Tiernan JP, Verghese ET, Nair A, Pathak S, Kim B, White J, et al. Systematic review and meta-analysis of cytokeratin 19-based one-step nucleic acid amplification versus histopathology for sentinel lymph node assessment in breast cancer. Br J Surg. Março de 2014;101(4):298–306. 8. Heilmann T, Mathiak M, Hofmann J, Mundhenke C, van Mackelenbergh M, Alkatout I, et al. Intra-operative use of one-step nucleic acid amplification (OSNA) for detection of the tumor load of sentinel lymph nodes in breast cancer patients. J Cancer Res Clin Oncol. Outubro de 2013;139(10):1649–55. 9. Chaudhry A, Williams S, Cook J, Jenkins M, Sohail M, Calder C, et al. The real-time intra-operative evaluation of sentinel lymph nodes in breast cancer patients using One Step Nucleic Acid Amplification (OSNA) and implications for clinical decision-making. Eur J Surg Oncol. Fevereiro de 2014;40(2):150–7.

Changes in the prevalence, incidence and residual risk for HIV and hepatitis C virus in Southern Brazilian blood donors since the implementation of NAT screening

Introduction Previous studies have shown high residual risk of transfusing a blood donation contaminated by human immunodeficiency virus (HIV) or hepatitis C virus (HCV) in Brazil and motivated the development of a Brazilian platform for simultaneous detection of both viruses by nucleic acid amplification test (NAT) denominated HIV/HCV Bio-Manguinhos/Fundação Oswaldo Cruz (FIOCRUZ). The objective of this study was to verify seroprevalence, incidence and residual risk for both viruses before and after the implementation of NAT. Methods Over 700,000 blood samples from all blood banks in the southern Brazilian State of Santa Catarina were analyzed during the period between January 2007 and July 2013. Results Compared with the period preceding the NAT screening, HIV prevalence increased from 1.38 to 1.58 per 1,000 donors, HIV incidence rate increased from 1.22 to 1.35 per 1,000 donor-years, and HIV residual risk dropped almost 2.5 times during the NAT period. For HCV, seroprevalence increased from 1.22 to 1.35 per 1,000 donors, incidence dropped from 0.12 to 0.06 per 1,000 donor-years, and residual risk decreased more than 3 times after the NAT implementation. Conclusions NAT reduced the duration of the immunologic window for HIV and HCV, thus corresponding to approximately 2.5- and 3-fold respective residual risk reductions.

The viral nature of Toddia França, 1912

Toddia França, 1912 under the light microscope occurs as inclusion corpuscles in the cytoplasm of erythrocytes of cold-blooded vertebrates sometimes accompanied by crystalloid bodies. Its position among the protozoans or the viruses has been discussed by some authors, but remained unclear. To elucidate this problem we studied Toddia from a Brazilian frog (Leptodactylus ocellatus) by electron microscopy. In the cytoplasm of the infected cells we found no protozoan, but rather virus-like particles often hexagonal in outline, averaging 195 nm excluding their two involving membranes, and presenting a central area of variable electron density. Particles at different stages of development were generally found around or on area lighter density than the cytoplasm. which resembled a virus synthesis site. At high magnification, the nuclear or cytoplasmic crystals allied to Toddia resembled the crystalline lattice of the inclusion bodies associated with the polyhedrosis viruses and poxviruses from insects, of the capsules of granulosis viruses and of other protein crystals in ultrathin sections. Cytochemical tests in Toddia corpuscles displayed exclusively the presence of deoxyribonucleic acid. These findings indicate that Toddia is not a protozoan and demonstrate that it is in all probability a viral inclusion corpuscle. Taking into account the nucleic acid type found in its structure (DNA) and the hexagonal shape usually shown in ultrathin sections by its component particles, which have a cytoplasmic site of synthesis and assembly, we tentatively relate Toddia with the so-called "Icosahedral Cytoplasmic Deoxyriboviruses". We believe that the present paper gives the first report of virus-like particles in L. ocellatus.

Burkholderia sartisoli sp. nov., isolated from a polycyclic aromatic hydrocarbon-contaminated soil.

A Gram-negative, rod-shaped, aerobic bacterium, designated strain RP007(T), was isolated from a polycyclic aromatic hydrocarbon-contaminated soil in New Zealand. Two additional strains were recovered from a compost heap in Belgium (LMG 18808) and from the rhizosphere of maize in the Netherlands (LMG 24204). The three strains had virtually identical 16S rRNA gene sequences and whole-cell protein profiles, and they were identified as members of the genus Burkholderia, with Burkholderia phenazinium as their closest relative. Strain RP007(T) had a DNA G+C content of 63.5 mol% and could be distinguished from B. phenazinium based on a range of biochemical characteristics. Strain RP007(T) showed levels of DNA-DNA relatedness towards the type strain of B. phenazinium and those of other recognized Burkholderia species of less than 30 %. The results of 16S rRNA gene sequence analysis, DNA-DNA hybridization experiments and physiological and biochemical tests allowed the differentiation of strain RP007(T) from all recognized species of the genus Burkholderia. Strains RP007(T), LMG 18808 and LMG 24204 are therefore considered to represent a single novel species of the genus Burkholderia, for which the name Burkholderia sartisoli sp. nov. is proposed. The type strain is RP007(T) (=LMG 24000(T) =CCUG 53604(T) =ICMP 13529(T)).

Rapid diagnostic tests for non-malarial febrile illness in the tropics.

The recent roll-out of rapid diagnostic tests (RDTs) for malaria has highlighted the decreasing proportion of malaria-attributable illness in endemic areas. Unfortunately, once malaria is excluded, there are few accessible diagnostic tools to guide the management of severe febrile illnesses in low resource settings. This review summarizes the current state of RDT development for several key infections, including dengue fever, enteric fever, leptospirosis, brucellosis, visceral leishmaniasis and human African trypanosomiasis, and highlights many remaining gaps. Most RDTs for non-malarial tropical infections currently rely on the detection of host antibodies against a single infectious agent. The sensitivity and specificity of host-antibody detection tests are both inherently limited. Moreover, prolonged antibody responses to many infections preclude the use of most serological RDTs for monitoring response to treatment and/or for diagnosing relapse. Considering these limitations, there is a pressing need for sensitive pathogen-detection-based RDTs, as have been successfully developed for malaria and dengue. Ultimately, integration of RDTs into a validated syndromic approach to tropical fevers is urgently needed. Related research priorities are to define the evolving epidemiology of fever in the tropics, and to determine how combinations of RDTs could be best used to improve the management of severe and treatable infections requiring specific therapy.

Extrachromosomal nucleic acids in bovine babesia

Two kinds of small extrachromosomal nucleic acid elements were found in the bovine babesias, Babesia bovis and B. bigemina. One element with an apparent size of 5.5 kilobase pairs (kbp) is a double stranded RNA related to virus like particles. Another molecule is a double stranded DNA with a molecular size of about 6.2 kbp. Southern blot comparison of restriction DNA fragments of the latter molecule, which is present in both B. bovis and B. bigemina is described.