Genotypic testing on HIV-1 DNA as a tool to assess HIV-1 co-receptor usage in clinical practice: results from the DIVA study group.

PURPOSE: We have developed a sequencing assay for determining the usage of the genotypic HIV-1 co-receptor using peripheral blood mononuclear cell (PBMC) DNA in virologically suppressed HIV-1 infected patients. Our specific aims were to (1) evaluate the efficiency of V3 sequences in B versus non-B subtypes, (2) compare the efficiency of V3 sequences and tropism prediction using whole blood and PBMCs for DNA extraction, (3) compare the efficiency of V3 sequences and tropism prediction using a single versus a triplicate round of amplification. RESULTS: The overall rate of successful V3 sequences ranged from 100 % in samples with >3,000 copies HIV-1 DNA/10(6) PBMCs to 60 % in samples with <100 copies total HIV-1 DNA /10(6) PBMCs. Analysis of 143 paired PBMCs and whole-blood samples showed successful V3 sequences rates of 77.6 % for PBMCs and 83.9 % for whole blood. These rates are in agreement with the tropism prediction obtained using the geno2pheno co-receptor algorithm, namely, 92.1 % with a false-positive rate (FPR) of 10 or 20 % and of 96.5 % with an FPR of 5.75 %. The agreement between tropism prediction values using single versus triplicate amplification was 98.2 % (56/57) of patients using an FPR of 20 % and 92.9 % (53/57) using an FPR of 10 or 5.75 %. For 63.0 % (36/57) of patients, the FPR obtained via the single amplification procedure was superimposable to all three FPRs obtained by triplicate amplification. CONCLUSIONS: Our results show the feasibility and consistency of genotypic testing on HIV-1 DNA tropism, supporting its possible use for selecting patients with suppressed plasma HIV-1 RNA as candidates for CCR5-antagonist treatment. The high agreement between tropism prediction by single and triple amplification does not support the use of triplicate amplification in clinical practice.

Drug-resistance development differs between HIV-1-infected patients failing first-line antiretroviral therapy containing nonnucleoside reverse transcriptase inhibitors with and without thymidine analogues.

OBJECTIVES: We evaluated the emergence of drug resistance in patients failing first-line regimens containing one nonnucleoside reverse transcriptase inhibitor (NNRTI) administered with zidovudine (ZDV) + lamivudine (the ZDV group) or non-thymidine analogues (non-TAs) (tenofovir or abacavir, + lamivudine or emtricitabine; the non-TA group). METHODS: Three hundred HIV-1-infected patients failing a first-line NNRTI-containing regimen (nevirapine, n = 148; efavirenz, n = 152) were included in the analysis. Virological failure was defined as viraemia ≥ 400 HIV-1 RNA copies/mL for the first time at least 6 months after starting the NNRTI-based regimen. For each patient, a genotypic resistance test at failure was available. The presence of drug-resistance mutations in HIV-1 reverse transcriptase was evaluated by comparing patients treated with NNRTI + zidovudine + lamivudine vs. those treated with NNRTI + non-TA. RESULTS: A total of 208 patients were failing with NNRTI + zidovudine + lamivudine and 92 with NNRTI + non-TA. No significant differences were observed between the non-TA group and the ZDV group regarding the time of virological failure [median (interquartile range): 12 (8-25) vs. 13 (9-32) months, respectively; P = 0.119] and viraemia [median (interquartile range): 4.0 (3.2-4.9) vs. 4.0 (3.3-4.7) log10 copies/mL, respectively; P = 0.894]. Resistance to reverse transcriptase inhibitors (RTIs) occurred at a significant lower frequency in the non-TA group than in the ZDV group (54.3 vs. 75.5%, respectively; P = 0.001). This difference was mainly attributable to a significantly lower prevalence of NNRTI resistance (54.3 vs. 74.0%, respectively; P = 0.002) and of the nucleoside reverse transcriptase inhibitor (NRTI) mutation M184V (23.9 vs. 63.5%, respectively; P < 0.001) in the non-TA group compared with the ZDV group. As expected, the mutation K65R was found only in the non-TA group (18.5%; P < 0.001). CONCLUSIONS: At first-line regimen failure, a lower prevalence of RTI resistance was found in patients treated with NNRTI + non-TA compared with those treated with NNRTI + zidovudine + lamivudine. These results confirm that the choice of backbone may influence the prevalence of drug resistance at virological failure.

Allele-sharing statistics using information on family history.

When conducting genetic studies for complex traits, large samples are commonly required to detect new genetic factors. A possible strategy to decrease the sample size is to reduce heterogeneity using available information. In this paper we propose a new class of model-free linkage analysis statistics which takes into account the information given by the ungenotyped affected relatives (positive family history). This information is included into the scoring function of classical allele-sharing statistics. We studied pedigrees of affected sibling pairs with one ungenotyped affected relative. We show that, for rare allele common complex diseases, the proposed method increases the expected power to detect linkage. Allele-sharing methods were applied to the symptomatic osteoarthritis GARP study where taking into account the family-history increased considerably the evidence of linkage in the region of the DIO2 susceptibility locus.

Prevalence of mutations and determinants of genotypic resistance to etravirine (TMC125) in a large Italian resistance database (ARCA).

OBJECTIVES: To evaluate whether etravirine (TMC125) might be effective in patients failing therapy with current nonnucleoside reverse transcriptase inhibitors (NNRTIs), we analysed the prevalence of TMC125 mutations and the possible determinants of genotypic resistance to this drug among sequences reported to a large database in Italy [Antiretroviral Resistance Cohort Analysis (ARCA)]. METHODS: We analysed the prevalence of TMC125 resistance-associated mutations (RAMs) and the TMC125 weighted genotypic score (WGS) together with the determinants of genotypic resistance. A total of 5011 sequences from 2955 patients failing NNRTI therapy were evaluated. RESULTS: Among the sequences in ARCA, 68% had at least one and 9.8% at least three TMC125 RAMs, whereas 31% had a WGS>2. Frequent RAMs were Y181C, G190A, K101E and A98G, whereas V179F, Y181V and G190S appeared in <5% of sequences. Multivariate analysis revealed a higher risk of developing at least three TMC125 RAMs associated with both nevirapine and efavirenz exposure, whereas CD4 counts > or = 200 cells/microL retained their protective effect. An increased risk of WGS>2 was linked to higher HIV RNA values (maximum risk at >5 log(10) copies/mL) and nevirapine exposure; CD4 counts > or = 200 cells/microL were protective. CONCLUSIONS: The prevalence of TMC125 resistance mutations in the ARCA cohort was 68%. The DUET studies showed that at least three TMC125-associated mutations were required to impair the efficacy of the drug and Y181C/V, V179F and G190S had the greatest effect on response. The prevalence of these mutations among the patients examined in our study was low. However, WGS>2 was found for one-third of our sequences. Previous nevirapine exposure was associated with an increased risk of having WGS>2 (adjusted odds ratio 1.76).

Score test for age at onset genetic linkage analysis in selected sibling pairs.

A new score statistic is derived, which uses information from registries (age-specific incidences) and family studies (sib-sib marginal correlation) to weight affected sibling pairs according to their age at onset. Age at onset of sibling pairs is modelled by a gamma frailty model. From this model we derive a bivariate survival function, which depends on the marginal survival and on the marginal correlation. The score statistic for linkage is a classical nonparametric linkage (NPL) statistic where the identical by descent sharing is weighted by a particular function of the age at onset data. Since the statistic is based on survival models, it can also be applied to discordant and healthy sibling pairs. Simulation studies show that the proposed method is robust and more powerful than standard NPL methods. As illustration we apply the new score statistic to data from a breast cancer study.

Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data.

Single nucleotide polymorphisms (SNPs) are often determined using TaqMan real-time PCR assays (Applied Biosystems) and commercial software that assigns genotypes based on reporter probe signals at the end of amplification. Limitations to the large-scale application of this approach include the need for positive controls or operator intervention to set signal thresholds when one allele is rare. In the interest of optimizing real-time PCR genotyping, we developed an algorithm for automatic genotype calling based on the full course of real-time PCR data. Best cycle genotyping algorithm (BCGA), written in the open source language R, is based on the assumptions that classification depends on the time (cycle) of amplification and that it is possible to identify a best discriminating cycle for each SNP assay. The algorithm is unique in that it classifies samples according to the behavior of blanks (no DNA samples), which cluster with heterozygous samples. This method of classification eliminates the need for positive controls and permits accurate genotyping even in the absence of a genotype class, for example when one allele is rare. Here, we describe the algorithm and test its validity, compared to the standard end-point method and to DNA sequencing.

Abnormal scattergrams and cell population data generated by fully automated hematological analyzers: New tools for screening malaria infection?

INTRODUCTION: Malaria is a life-threatening infectious disease, which has been for long confined to specific endemic areas. Nevertheless, the recent increase in immigration flows from endemic regions and imported cases has reemphasized many diagnostic challenges in Western countries, thus paving the way to introduce rapid and accurate strategies for screening subjects with suspected Malaria infection. Therefore, the aim of this article was to describe our recent experience with Sysmex XN-module for rapid screening of subjects with suspected Malaria. METHODS: Fourteen patients admitted to the Emergency Department (Papa Giovanni XXIII Hospital Bergamo, Italy) with a clinical suspicion of Malaria infection were evaluated, along with 1047 control samples. The analysis of peripheral blood was performed with XN-module, and results were then compared to optical microscopy. RESULTS: Nine patients were positive to Plasmodim falciparum, 3 to Plasmodim vivax, one to Plasmodim ovale, and one to Plasmodim malarie. Characteristic abnormalities could be observed in both white blood cell differential (WDF) and white cell nucleated (WNR) scattergrams (sensitivity 0.64 and specificity 1.0) in 9 samples with parasites at gametocyte or schizos stage irrespective of Plasmodium species and parasitic index, while characteristic scattergram abnormalities could not be seen in the 5 samples containing only parasites at the trophozoites stage. In these cases, specific variations of some cell population data (CPD) could be recorded (sensitivity 1.00 and specificity 0.91). CONCLUSION: The peculiar abnormalities observed in CPDs, WDF, and WNR-scattergrams may raise a definite suspicion of Malaria infection. Further studies should then be planned for validating these preliminary findings and assessing whether these specific abnormalities may be incorporated in rapid and inexpensive Malaria diagnostic algorithms.

A locally adaptive statistical procedure (LAP) to identify differentially expressed chromosomal regions.

MOTIVATION: The systematic integration of expression profiles and other types of gene information, such as chromosomal localization, ontological annotations and sequence characteristics, still represents a challenge in the gene expression arena. In particular, the analysis of transcriptional data in context of the physical location of genes in a genome appears promising in detecting chromosomal regions with transcriptional imbalances often characterizing cancer. RESULTS: A computational tool named locally adaptive statistical procedure (LAP), which incorporates transcriptional data and structural information for the identification of differentially expressed chromosomal regions, is described. LAP accounts for variations in the distance between genes and in gene density by smoothing standard statistics on gene position before testing the significance of their differential levels of gene expression. The procedure smooths parameters and computes p-values locally to account for the complex structure of the genome and to more precisely estimate the differential expression of chromosomal regions. The application of LAP to three independent sets of raw expression data allowed identifying differentially expressed regions that are directly involved in known chromosomal aberrations characteristic of tumors. AVAILABILITY: Functions in R for implementing the LAP method are available at http://www.dpci.unipd.it/Bioeng/Publications/LAP.htm

Prevalence of etravirine (ETR)-RAMs at NNRTI failure and predictors of resistance to ETR in a large Italian resistance database (ARCA).

The prevalence of drug resistance associated with the failure of non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimens and the predictors of resistance to Etravirine (ETR) were assessed in 2854 subjects: 39 < 18 (paediatric) and 2815 ≥ 18 (adult) years old. These subjects failed to respond to their current NNRTI treatment, were three-class experienced and had been exposed to NNRTI for ≥3 months. A total of 1827 adult (64.9%) and 32 paediatric subjects (82.1%) harboured the virus with at least one ETR mutation. V179I, Y181C and G190A were the most frequent mutations in both groups. A significantly increased risk of ETR resistance with all three algorithms (Monogram (MGR) >3, Tibotec (TBT) >2 and enhanced MGR (ENH) ≥4) emerged in the paediatric population. Multivariate analysis revealed an increased risk of developing TBT >2 for NNRTI exposure, ENH ≥4 for NNRTI and EFV exposure in paediatric subjects; NVP exposure and higher (≥3.5 log10) HIV-RNA values for all three algorithms in adult subjects, whereas CD4 ≥ 200/µL appeared to be protective. The risk of being ETR resistant was more than doubled for paediatric vs. adult subjects, probably due to a more extensive use of NNRTI and an incomplete virological control.

Human immunodeficiency virus-1 B and non-B subtypes with the same drug resistance pattern respond similarly to antiretroviral therapy.

We analysed the 12-week virological response to protease inhibitor (PI) or non-nucleoside reverse transcriptase inhibitor (NNRTI) therapy in 1108 patients carrying B or non-B human immunodeficiency virus (HIV)-1 subtypes with matched resistance mutation patterns. Response rates were not significantly different for non-B and B subtypes stratified for treatment status (51.5% vs. 41.5% in naïve patients; 46.7% vs. 38.7% in experienced patients) or regimens (46.9% vs. 39.7% with PI; 56.7% vs. 40% with NNRTI). No difference in response was detected in patients harbouring B and non-B subtypes with any resistance profile. Further studies are advisable to fully test this approach on larger datasets.

Weighted statistics for aggregation and linkage analysis of human longevity in selected families: the Leiden Longevity Study.

Typically long-lived sibling pairs have been collected for linkage analysis of human longevity and information on life span of first-degree relatives is available to assess familial aggregation of life span. We propose a new weighted statistic for aggregation analysis, which tests for a relationship between a family history of excessive survival of the sibships of the long-lived pairs and the survival of their parents and their offspring. For linkage analysis, we derive a new weighted score statistic from a simple gamma frailty model, which assigns more weight to excessive long-lived pairs. We apply the methods to data from the Leiden Longevity Study, which consists of sibling pairs of age 90 years or above and their first-degree relatives. The pairs have been genotyped for microsatellite markers in a candidate region. Association was present between survival within the sibships and survival of the offspring, but not with the parental generation. For linkage analysis, weighting increased the value of the test statistic, but the result was not statistically significant. About the methods we conclude that the statistic for aggregation provides insight into clustering of life span and the statistic for linkage provides a new tool to include demographic information into the analysis.

Suplementação com gordura protegida para vacas de corte desmamadas precocemente mantidas em pastagem natural / Protected fat supplementation for early weaned beef cows maintained in nature pasture

Avaliaram-se os desempenhos produtivo e reprodutivo de vacas de corte, bem como o desempenho de seus bezerros, de acordo com os tratamentos alimentares: PRE: suplementação com gordura protegida (GP) 45 dias antes do parto; PREPOS: suplementação com GP 45 dias antes do parto e 63 dias pós-parto; POS: suplementação com GP 63 dias pós-parto; PN: sem suplementação. O desempenho produtivo das vacas não foi influenciado pelo manejo alimentar (P>0,05), exceto para o escore da condição corporal (ECC) no final do período de acasalamento, que foi mais baixo para as vacas do PRE e do PREPOS, sendo que esta última apresentou ECC semelhante ao das vacas do POS e do PN. O intervalo entre partos foi menor para as vacas do tratamento PREPOS - 376 dias -, não diferindo das vacas do PN - 383 dias. As vacas do PREPOS desmamaram 4,4 por cento mais quilos de bezerro para cada 100kg de vaca ao parto - 22,6kg - do que as vacas do PRE e do POS - 21,6kg e 21,6kg, respectivamente - e 8,4 por centomais quilos de bezerro para cada 100kg de vaca ao parto do que as vacas mantidas em pastagem nativa - 20,7kg. A suplementação com gordura protegida durante os períodos pré e/ou pós-parto não afeta o desempenho de vacas e bezerros...

The objective of this work was to evaluate the productive and reproductive performance of beef cows, as well as the performance of their calves according to the following dietary treatments: PRE: supplemented with protected fat (PF) during 45 days prepartum; PREPOS: supplemented with PF during 45 days prepartum and 63 days postpartum; POS: supplemented with PF during 63 days postpartum; PN: without supplementation. The productive performance of cows was not influenced by feed management (P>0.05), except for body condition score (BCS), which was lower for PRE and PREPOS cows at the end of mating season, with the latter cows having similar BCS POS and PN. The calving interval (CI) was shorter for cows supplemented in PREPOS - 376 days -, and did not differ in cows maintained in PN - 383 days. Supplemented PREPOS cows weaned 4.4 percent more pounds of calf per 100kg of cow at calving - 22.6kg - than the PRE and POS cows - 21.6kg and 21.6kg, respectively - and 8,4 percent more pounds of calf per 100 of cow at calving than the cows maintained in native pasture - 20.7kg. The fat protected supplementation during pre and/or postpartum periods did not affect the performance of cows and calves...

Metabólitos sanguíneos de vacas de corte suplementadas ou não com sais de cálcio de ácidos graxos durante o período pré e/ou pós-parto / Blood metabolites from beef cows supplemented or not with calcium salts of fatty acids during pre and/or postpartum period

Avaliou-se a concentração de metabólitos sanguíneos de vacas de corte mantidas em pastagem natural recebendo suplementação com sais de cálcio de ácidos graxos (SCAG) durante 45 dias antes do parto (PRE), suplementação com SCAG durante 45 dias antes do parto e 63 dias pós-parto (PREPOS), suplementação com SCAG durante 63 dias pós-parto (POS) e de vacas não suplementadas (PN). As coletas de sangue foram realizadas em média 64 dias antes do parto, e aos 21, 42 e 63 dias pós-parto. Não ocorreu interação significativa entre tratamentos e períodos. As concentrações plasmáticas de β-hidroxibutirato (βHB), triglicerídeos (TRIG), colesterol, glicose e ureia não foram afetadas significativamente pela suplementação de gordura protegida. A análise de regressão mostrou queda linear significativa da concentração de TRIG no sangue com o aumento da produção de leite (PL) para as vacas do tratamento PN (TRIG = 23,10 - 2,18*PL, R² = 0,31) e efeito quadrático para as vacas do PRE (TRIG = 6,54 - 1,75*PL + 0,30*PL², R² = 0,62). Nos animais dos tratamentos POS e PREPOS, não houve efeito da produção de leite sobre a concentração de TRIG, indicando que a suplementação durante o período de produção de leite auxilia na manutenção de um balanço energético positivo. As concentrações de colesterol plasmático aumentaram, e as de triglicerídeos e ureia diminuíram linearmente até o final do experimento.

The study evaluated the blood metabolites of beef cows maintained on native pasture supplemented with calcium salts of fatty acids (CSFA) during 45 days pre-partum (PRE); supplemented with CSFA during 45 days pre-partum and 63 days post-partum (PREPOS); supplemented with CSFA during 63 days post-partum (POS) and cows not supplemented (PN): without supplementation. Blood samples were taken 64 days pre-partum, and at 21, 42 and 63 days post-partum. No significant interaction was observed between treatment and period. Plasma concentrations of β-hydroxybutyrate (βHB), triglycerides, cholesterol, glucose and urea were not affected by protected fat supplementation. The regression analysis showed significant linear decline of blood triglycerides (TRIG) concentration with the increase of milk production (PL) for PN cows (TRIG= 23.10 - 2.18*PL, R²= 0.31) and quadratic effect for PRE cows (TRIG= 6.54 - 1.75*PL + 0.30*PL², R²= 0.62). For cows submitted to the POS and PREPOS treatments there was no effect of milk production on blood triglycerides concentration, indicating that CSFA during milk production aids the maintenance of a positive energetic balance. The concentration of plasma cholesterol increased while the concentrations of triglycerides and urea decreased linearly until the end of the experiment.

Características dos componentes não integrantes da carcaça de novilhos superjovens da raça Devon, terminados em diferentes sistemas de alimentação / Characteristics of non-integrant components of carcass of Devon young steers, finished in different feeding systems

O objetivo do estudo foi avaliar as características dos componentes não integrantes da carcaça de novilhos Devon terminados em confinamento (CONF), em pastagem de clima temperado (pastagem de azevém - Lolium multiflorum Lam - PTEM) ou em pastagem de clima tropical (associação de pastagem de milheto - Pennisetum americanum (L.) Leeke - e capim-papuã - Bracharia plantaginea - PTRO). Os novilhos, ao início da terminação, estavam com 320kg e 15 meses de idade. Os animais confinados foram alimentados com relação volumoso:concentrado de 60:40; o volumoso era constituído de silagem de milho, e o concentrado de farelo de trigo, milho e minerais. Os animais foram abatidos com pesos semelhantes de 388,3; 386,7 e 375,8kg no CONF, na PTEM e na PTRO, respectivamente. Os animais da PTRO apresentaram maior (P<0,10) rendimento de carcaça quente (RCQ) relativo a 100kg de peso corporal vazio (RCQPCV) do que os da PTEM, 64,6 versus 62,6 por cento, e os do CONF apresentaram RCQPCV intermediário, 63,7 por cento. Os pesos absolutos do fígado, 5,22; 4,43 e 3,87kg, do conjunto dos órgãos internos, 12,81; 11,37 e 10,83kg, do rúmen-retículo, 7,62; 6,54 e 6,06kg, da gordura do coração, 1,26; 0,65 e 0,30kg, e dos intestinos, 9,97; 7,13 e 7,49kg, foram mais altos (P<0,05) nos animais da PTEM, em relação aos do CONF e da PTRO, respectivamente. A mesma ordem de grandeza ocorreu com os pesos relativos desses órgãos. A PTRO e o CONF originaram animais com maior (P<0,05) peso de conteúdo gastrintestinal em relação à PTEM, respectivamente, 60,27; 55,32 e 41,21kg. O CONF proporcionou animais com pesos absolutos mais elevados (P<0,05) do omaso, 5,17kg, em relação aos da PTEM, 3,70kg, e este peso foi intermediário nos animais da PTRO, 4,77kg. A mesma ordem de grandeza ocorreu com os pesos relativos do omaso, 1,61; 1,12 e 1,54 por cento.

The non-integrant components of carcass of Devon steers were evaluated. Animals were finished in feedlot (CONF), winter pasture (pasture of ryegrass - Lolium multiflorum Lam -PTEM), or tropical pasture (association of millet pasture - Pennisetum americanum (L.) Leeke - and Alexander Grass - Brachiaria plantaginea - PTRO). At the beginning of finishing, the average weight of steers was 320kg and age was 15 months. The roughage:concentrate ratio of CONF was 60:40. The animals were slaughtered at similar weights of 388.3, 386,7, and 375.8 for CONF, PTEM, and PTRO, respectively. The PTRO animals showed higher hot carcass dressing (HCD) percentage relative to 100kg of empty body weight (HCDEBW) in comparison to PTEM (64.6 versus 62.6 percent), and CONF animals showed intermediary value for HCDEBW (63.7 percent). The absolute weights of liver, 5.22, 4.43, and 3.87kg; total weights of all internal organs, 12.81, 11.37, and 10.83kg; rumen-reticulum, 7.62, 6.54, and 6.06kg; heart fat, 1.26, 0.65, and 0.30kg; and intestines, 9.97, 7.13, and 7.49kg, were higher (P<0.05) in PTEM animals than in CONF and PTRO animals, respectively. The same differences were observed concerning relative weights of the same organs. PTRO and CONF animals showed higher (P<0.05) gastrointestinal content than PTEM animals, 60.27, 55.32, and 41.21kg, respectively. Feedlot finished animals exhibited higher absolute weight of omasum, 5.17kg, than PTEM animals, 3.70kg, and PTRO animals presented intermediary value, 4,77kg. The same order was observed concerning relative weigh of the omasum, 1.61, 1.12, and 1.54 percent.

Comparative evaluation of two commercial amplification assays for direct detection of Mycobacterium tuberculosis complex in respiratory specimens.

Two commercial assays detecting the presence of Mycobacterium tuberculosis complex in clinical specimens by rRNA target amplification (Gen-Probe Amplified M. tuberculosis Direct Test [AMTD]) and PCR (Amplicor) were evaluated. The tests were applied to 327 digested, decontaminated respiratory specimens collected from 236 patients. Results were compared with those of acid-fast staining and culture. The combination of culture and clinical diagnosis was considered the "gold standard." A total of 60 specimens were collected from 27 patients with a diagnosis of pulmonary tuberculosis. Thirteen of these specimens were from patients receiving standard antituberculosis therapy and therefore were not included in the comparison. Of the remaining 47 specimens, 33 were smear positive, 40 were culture positive, 45 were AMTD positive, and 39 were Amplicor positive. After resolution of discrepant results, the overall sensitivities, specificities, and positive and negative predictive values were 77, 100, 100, and 95 for staining; 87, 100, 100, and 97.4 for culture; 95.9, 98.9, 94, and 99.2 for AMTD; and 85.4, 99.6, 97.9, and 97.1 for Amplicor, respectively. Agreement between AMTD and Amplicor assay results was 96.8%. It is concluded that although both nucleic acid amplification methods are rapid and specific for the detection of M. tuberculosis complex in respiratory specimens, AMTD appeared to be more sensitive than Amplicor.

Financial impact in the Italian Health Service of laparoscopic versus laparotomic surgery for the treatment of ovarian cysts.

To assess the cost of two procedures for the removal of ovarian cysts, 200 pre-menopausal women were recruited for the surgical removal of ovarian cysts by laparoscopy (n = 100) and laparotomy (n = 100) according to case-control criteria. Patients operated by laparoscopy (mean age +/- SD 32.22 +/- 9.98 years) and laparotomy (mean age +/- SD 29.57 +/- 6.62 years) for ovarian cysts (mean diameters +/- SD 4.98 +/- 3.62 and 4.83 +/- 2.78 cm) had a post-surgical hospital stay of 3.12 +/- 0.41 and 7.25 +/- 1.08 days (P < 0.001) respectively. The total rate of complications occurring in patients operated by laparoscopy was 9 versus 53% (P < 0.001) of those operated by laparotomy; body temperature > 38 degrees C was recorded in 52/100 of patients operated by laparotomy versus 6/100 of those operated by laparoscopy. The mean cost for each pure surgical treatment performed by laparoscopy was US $498.17 versus US $642.47 when it was performed by laparotomy (P < 0.001). The laparoscopic surgical approach is more expensive in the first 36 operations, thereafter becoming cheaper. The mean of the entire overall expenditure was US $1142.08 and US $2138.72 for laparoscopy and laparotomy (P < 0.001) respectively. The entire expenditure for laparoscopy is higher than laparotomy only until eight operations. In conclusion, laparoscopy versus laparotomy has resulted in a saving of US $14,429.3 for 100 operations while the saving on entire costs was US $99,664.8.

Mycobacterium malmoense in Italy: the modern Norman invasion?

The isolation of Mycobacterium malmoense has for a long time been restricted to few countries of Northern Europe; reports from countries other than Sweden, Great Britain and Finland are rare and the first Italian case report has been published in 1995. Since 1988, however, fifteen strains of M. malmoense have been isolated in Italy, eleven of which in the last two years; of these, ten appeared clinically significant on the basis of medical records. The susceptibility of the strains and the role of high performance liquid chromatography of cell wall mycolic acids for a reliable identification are discussed.

Comparative evaluation of the new gen-probe Mycobacterium tuberculosis amplified direct test and the semiautomated abbott LCx Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis complex in respiratory and extrapulmonary specimens.

Two commercial assays that detect Mycobacterium tuberculosis complex (MTB) in clinical specimens by rRNA target amplification (AMTDII) and ligase chain reaction (LCx) were evaluated. The tests were applied to 457 respiratory (n = 273) and extrapulmonary (n = 184) specimens collected from 357 patients. The results were compared with those of acid-fast staining and culture. The combination of culture and clinical diagnosis was considered to be the "gold standard." Seventy specimens were from patients with pulmonary tuberculosis and 28 specimens were from patients with extrapulmonary tuberculosis. After resolution of discrepant results, the overall sensitivities, specificities, and positive and negative predictive values for respiratory specimens were 92.8, 99.4, 98.5, and 97%, respectively, for AMTDII and 75.7, 98.8, 96.4, and 90.5%, respectively, for LCx. With extrapulmonary specimens, the overall sensitivities, specificities, and positive and negative predictive values were 78.6, 99.3, 95.6, and 96.2%, respectively, for AMTDII and 53.6, 99.3, 93.7, and 92.1%, respectively, for LCx. The level of agreement between AMTDII and LCx assay results was 78.2%. We conclude that although both nucleic acid amplification methods are rapid and specific for the detection of MTB in clinical specimens, AMTDII is significantly more sensitive than LCx with both respiratory (P = 0.005) and extrapulmonary (P = 0.048) specimens.