Results of An International Study of Quantitative BCR/ABL Assays Using Defined RNA Samples

Molecular monitoring of BCR/ABL transcripts by real time quantitative reverse transcription PCR (qRT-PCR) is an essential technique for clinical management of patients with BCR/ABL-positive CML and ALL. Though quantitative BCR/ABL assays are performed in hundreds of laboratories worldwide, results among these laboratories cannot be reliably compared due to heterogeneity in test methods, data analysis, reporting, and lack of quantitative standards. Recent efforts towards standardization have been limited in scope. Aliquots of RNA were sent to clinical test centers worldwide in order to evaluate methods and reporting for e1a2, b2a2, and b3a2 transcript levels using their own qRT-PCR assays. Total RNA was isolated from tissue culture cells that expressed each of the different BCR/ABL transcripts. Serial log dilutions were prepared, ranging from 100 to 10-5, in RNA isolated from HL60 cells. Laboratories performed 5 independent qRT-PCR reactions for each sample type at each dilution. In addition, 15 qRT-PCR reactions of the 10-3 b3a2 RNA dilution were run to assess reproducibility within and between laboratories. Participants were asked to run the samples following their standard protocols and to report cycle threshold (Ct), quantitative values for BCR/ABL and housekeeping genes, and ratios of BCR/ABL to housekeeping genes for each sample RNA. Thirty-seven (n=37) participants have submitted qRT-PCR results for analysis (36, 37, and 34 labs generated data for b2a2, b3a2, and e1a2, respectively). The limit of detection for this study was defined as the lowest dilution that a Ct value could be detected for all 5 replicates. For b2a2, 15, 16, 4, and 1 lab(s) showed a limit of detection at the 10-5, 10-4, 10-3, and 10-2 dilutions, respectively. For b3a2, 20, 13, and 4 labs showed a limit of detection at the 10-5, 10-4, and 10-3 dilutions, respectively. For e1a2, 10, 21, 2, and 1 lab(s) showed a limit of detection at the 10-5, 10-4, 10-3, and 10-2 dilutions, respectively. Log %BCR/ABL ratio values provided a method for comparing results between the different laboratories for each BCR/ABL dilution series. Linear regression analysis revealed concordance among the majority of participant data over the 10-1 to 10-4 dilutions. The overall slope values showed comparable results among the majority of b2a2 (mean=0.939; median=0.9627; range (0.399 - 1.1872)), b3a2 (mean=0.925; median=0.922; range (0.625 - 1.140)), and e1a2 (mean=0.897; median=0.909; range (0.5174 - 1.138)) laboratory results (Fig. 1-3)). Thirty-four (n=34) out of the 37 laboratories reported Ct values for all 15 replicates and only those with a complete data set were included in the inter-lab calculations. Eleven laboratories either did not report their copy number data or used other reporting units such as nanograms or cell numbers; therefore, only 26 laboratories were included in the overall analysis of copy numbers. The median copy number was 348.4, with a range from 15.6 to 547,000 copies (approximately a 4.5 log difference); the median intra-lab %CV was 19.2% with a range from 4.2% to 82.6%. While our international performance evaluation using serially diluted RNA samples has reinforced the fact that heterogeneity exists among clinical laboratories, it has also demonstrated that performance within a laboratory is overall very consistent. Accordingly, the availability of defined BCR/ABL RNAs may facilitate the validation of all phases of quantitative BCR/ABL analysis and may be extremely useful as a tool for monitoring assay performance. Ongoing analyses of these materials, along with the development of additional control materials, may solidify consensus around their application in routine laboratory testing and possible integration in worldwide efforts to standardize quantitative BCR/ABL testing.

Infective stages of Leishmania in the sandfly vector and some observations on the mechanism of transmission

Infective stages of Leishmania (Leishmania) amazonensis, capable of producing amastigote infections in hamster skin, were shown to be present in the experimentally infected sandfly vector Lutzomyia flaviscutellata 15, 25, 40, 49, 70, 96 and 120 hours after the flies had received their infective blood-meal. Similarly, infective stages of Leishmania (L.) chagasi were demonstrated in the experimentally infected vector Lu. longipalpis examined 38, 50, 63, 87, 110, 135, 171 and 221 hours following the infective blood-meal, by the intraperitoneal inoculation of the flagellates into hamsters. The question of whether or not transmission by the bite of the sandfly is dependent on the presence of [quot ]metacyclic[quot ] promastigotes in the mouthparts of the vector is discussed.

The transmission of suprapylarian Leishmania by bite of experimentally infected sand flies (Diptera: Psychodidae)

Lutzomyia furcata transmitted Leishmania chagasi to a hamster 10 days after being experimentally fed on an infected spleen. An individual female Psychodopygus carrerai carrerai that had fed on a hamster lesion caused by Leishmania mexicana amazonensis transmitted this parasite 6 days later to another hamster. Transmission electron microscopy of this fly's head revealed a small number of degenerate promastigotes in the foregut, but only a few were attached.

Evolution of insulin sensitivity and its variability in out-of-hospital cardiac arrest (OHCA) patients treated with hypothermia.

INTRODUCTION: Therapeutic hypothermia (TH) is often used to treat out-of-hospital cardiac arrest (OHCA) patients who also often simultaneously receive insulin for stress-induced hyperglycaemia. However, the impact of TH on systemic metabolism and insulin resistance in critical illness is unknown. This study analyses the impact of TH on metabolism, including the evolution of insulin sensitivity (SI) and its variability, in patients with coma after OHCA. METHODS: This study uses a clinically validated, model-based measure of SI. Insulin sensitivity was identified hourly using retrospective data from 200 post-cardiac arrest patients (8,522 hours) treated with TH, shortly after admission to the intensive care unit (ICU). Blood glucose and body temperature readings were taken every one to two hours. Data were divided into three periods: 1) cool (T <35°C); 2) an idle period of two hours as normothermia was re-established; and 3) warm (T >37°C). A maximum of 24 hours each for the cool and warm periods was considered. The impact of each condition on SI is analysed per cohort and per patient for both level and hour-to-hour variability, between periods and in six-hour blocks. RESULTS: Cohort and per-patient median SI levels increase consistently by 35% to 70% and 26% to 59% (P <0.001) respectively from cool to warm. Conversely, cohort and per-patient SI variability decreased by 11.1% to 33.6% (P <0.001) for the first 12 hours of treatment. However, SI variability increases between the 18th and 30th hours over the cool to warm transition, before continuing to decrease afterward. CONCLUSIONS: OCHA patients treated with TH have significantly lower and more variable SI during the cool period, compared to the later warm period. As treatment continues, SI level rises, and variability decreases consistently except for a large, significant increase during the cool to warm transition. These results demonstrate increased resistance to insulin during mild induced hypothermia. Our study might have important implications for glycaemic control during targeted temperature management.

Eimeria vitellini n. sp. (Apicomplexa: eimeriidae) from the brazilian toucan Rhamphastos vitellinus vitellinus Lichtenstein (Aves: Picicformes: Rhamplastidae)

Eimeria vitellini n. sp. is described from the faeces of the Rhamphastos v. vitellinus. Oocysts broadly ellipsoidal to oval (egg-shaped), 22,6 x 18.3 (20.0-25.0 x 16.3-22.5) micronm, shapeindex (length/width) 1.2 (1.1-1.1). Oocyst wall a single colourless layer about 0.5 micronm thick, becoming thinner at one ectremity, at which point the oocyst usually ruptures. No oocyst residuum, but 1 or 2 small polr bodies of about 1.0-1.5 x 0.5-1.0 micronm. Sporocysts ellongated ellipsoid (pearshaped), 14.3 x 7.5 (13.8-15.0 x 6.9-7.5) micronm, shape-index (1.9 (1.8-2.0), with a thin colourless wall bearing a very delicate Stieda body: a conspicuous sub-Stieda body is present. Sporozoites with anterior and posterior regractile bodies and strongly recurved around a bulky, compact sporocyst residuum composed of relatively fine granules and spherules.

Eimeria species (Apicomplexa: Eimeriidae) of podocnemis expansa (Schweigger) and geochelone denticulata (LINN.) from Amazonian Brazil (Reptilia: Chelonia)

Eimeria lagunculata, Eimeria mammiformis and Eimeria podocnemis n. spp., are described from the faeces of the fresh-water turtle Podocnemis expansa, in Pará State, north Brasil. Oocysts of E. lagunculata are ellipsoidal, 19.2 x 12.8 (17.0-20.7 x 11.8-14.1) mum, shape-index (= length/ width) 1.5 (1.4-1.7). Oocyst wall about 0.5-0.7 mum thick, with a prominent stopper-like micropyle at one pole. No oocyst residuum and no polar body. Sporocysts elongate ellipsoidal, 11.0 x 5.4 (10.4-11.8 x 5.2-6.0) mum, shape-index 2.0 (1.8-2.1): no Stieda body. A compact, ellipsoidal sporocyst residuum lies between the two sporozoites, which possess a posterior and an anterior refractile body. Oocysts of E. mammiformis broadly ellipsoidal, 30.0 x 19.4 (23.0-37.0 x 16.3-21.5) mum, shape-index 1.5 (1.1-1.9). Oocyst wall about 0.7 mum thick, with a prominent micropyle: no oocyst residuum and rarely a single polar body. Sporocysts ellipsoidal, 15.3 x 7.9 (14.8-17.0 x 7.4-9.6) mum, shape-index 2.0 (1.8-2.2), with a tiny Stieda body. Sporocyst residuum bulky, ellipsoidal: sporozoites with two conspicuous refractile bodies. E. podocnemis has broadly ellipsoidal oocysts, 17.0 x 12.8 (14.8-19.2 x 11.8-14.1) mum, shape-index 1.3 (1.1-1.4). Oocyst wall about 0.5-0.7 mum thick, with no micropyle. No oocyst residuum, but always a single polar body. Sporocysts ellipsoidal, 9.7 x 5.2 (8.9-10.4 x 4.4-6.0) mum, shape-index 1.9 (1.6-2.0), with no Stieda body. Sporocyst residuum bulky, ellipsoidal: sporocysts with 2 refractile bodies. Eimeria carinii n. sp., is recorded from the tortoise Geochelone denticulata, also from Pará. Oocyst wall about 1.2 mum thicl. No micropyle. Oocyst residuum limited to a number (about 10-20) of scattered granules: no polar body. Sporocysts broadly ellipsoidal, and with no Stieda body: they measure 8,8 x 7.3 (8.0-9.0 x 7.0-7.5) mum, shape-index 1.2 (1.1-1.3). Sporocyst residuum bulky, spherical to ellipsoidal: sporozoites possess both posterior and anterior refractile bodies.

DNA probes for distinguishing Psychodopygus wellcomei from Psychodopygus complexus (Diptera: Psychodidae)

Genomic DNA fragments from males of Psychodopygus wellcomei were isolated and shown to be useful as sensitive diagnostic probles for positively separting individuals of this species from those of Ps. complexus. These two members of the Ps. squamiventris series are found sympatrically in foci of cutaneous leishmaniasis in the hill forests of southern Pará State. Of the two species, only Ps. welcomei is thought to be an important vector of Leishmania braziliensis sensu stricto, buth this is based on circumstantial evidence because of the difficulties of identifying female sandflies wothin the series. The diagnostic probes were isolated from a library of Ps. wellcomei built by ligationg short fragments of Sau 3A-resistricted, genomic DNA into the plasmid vector PUC 18. Differential screening of 1316 library clones with total genomic DNA of Ps. Wellcomei and Ps. complexus identified 5 recombinants, with cross-hybridizing inserts of repetitive DNA, that showed strong specificity for Ps. wellcomei. As little as 0.4% of the DNA extracted from an individual sandfly (=ca. 0.5 namograms) was specifically detected. The diagnostic probes were used to identify as Ps. wellcomei a wild-caught female sandfly found infected with L. braziliensis s.s., providing only the second positive association between these two species.