Molecular detection of Aspergillus in respiratory samples collected from patients at higher risk of chronic pulmonary aspergillosis.

OBJECTIVE: Aspergillosis diagnosis depends on the detection of Aspergillus in biological samples ─ usually using cultural and immunoenzyme techniques ─ but their sensitivity and specificity varies. We aimed to study the prevalence of Aspergillus in patients at higher risk of chronic pulmonary aspergillosis (i.e., HIV-infected patients and individuals with active or previous tuberculosis), and to determine the potential role of molecular approaches to increase detection of Aspergillus in respiratory samples. METHODS: The DNA extracted from 43 respiratory samples that had been previously analyzed by immunoenzyme and/or cultural techniques was amplified by real-time multiplex PCR, and the results of these methods were compared. We also sequenced the ITS1 region and the calmodulin gene in 10 respiratory samples to perform a pilot metagenomic study to understand the ability of this methodology to detect potential pathogenic fungi in the lung mycobiome. RESULTS: Real-time Aspergillus PCR test exhibited a higher positivity rate than the conventional techniques used for aspergillosis diagnosis, particularly in individuals at risk for chronic pulmonary aspergillosis. The metagenomic analysis allowed for the detection of various potentially pathogenic fungi. CONCLUSIONS: Molecular techniques, including metagenomics, have great ability to detect potentially pathogenic fungi rapidly and efficiently in human biological samples.

Insights on catheter-related bloodstream infections: a prospective observational study on the catheter colonization and multidrug resistance.

BACKGROUND: Central venous catheter-related bloodstream infection (CRBSI) is a huge public health concern with considerable impact on mortality and health costs. AIM: A three-year observational study enrolling three tertiary hospitals located in Lisbon, Portugal, was designed to identify the major aetiological agents of CRBSI, their ability to colonize central venous catheters and their antimicrobial resistance profiles. METHODS: Aetiological agents of CRBSI were identified by Vitek 2. Whole-genome sequencing was used to confirm CRBSI by the most prevalent aetiological agents and characterize their resistome. Central venous catheter colonization (namely by biofilm assembly) was monitored by scanning electron microscopy. FINDINGS: Staphylococci were the most prevalent causative agent (36/58, 62.0%), with S. aureus and coagulase-negative S. epidermidis accounting for 24.1% and 36.2% of CRBSIs, respectively. Fifty-nine of 72 staphylococci isolates were meticillin resistant. Comparative genomic analysis of central venous catheters/haemoculture pairs of isolates revealed genomic matches for 35 of 36 pairs and a good correlation between antibiotic susceptibility phenotype and the presence of antimicrobial resistance genetic determinants. Biofilms were present on 48.6% of the central venous catheters; nevertheless, no statistically significant association was established between biofilm assembly and CRBSI, and the presence/absence of ica operon and agr groups did not correlate with biofilm phenotypes, highlighting the need for further studies to elucidate biofilms' role on this healthcare-associated infection. CONCLUSION: Whole-genome sequencing was shown to be a valuable tool to confirm CRBSI. Although more than 42.3% of the central venous catheters were colonized by staphylococci, no statistically significant association was found between CRBSI and biofilms.

Nontuberculous Mycobacteria in Portugal: Trends from the last decade.

INTRODUCTION AND OBJECTIVES: Nontuberculous mycobacteria (NTM) are opportunistic human pathogens found in the environment. The transmission seems to be associated with inhalation of aerosol droplets, ingestion or trauma events. Recent studies indicate that NTM disease is increasing worldwide, however, the true clinical impact of NTM infections is difficult to determine due to challenges in discriminating between disease and colonization as they are ubiquitous in the environment. In addition, understanding the epidemiology of NTM is difficult and has not yet been established. In this work, we used a country NTM representative collection from the National Reference Laboratory for Tuberculosis (NRL-TB) of the National Institute of Health (INSA), to characterize the circulation trends of NTM species in Portugal and the most affected regions, contributing to a better understanding of the NTM epidemiology. MATERIAL AND METHODS: We conducted a nationwide retrospective study where all individuals with positive NTM cultures at the NRL-TB of the INSA from 2014 to December 2020 were included. Positive cultures were identified using GenoType Mycobacterium CM/AS® (Hain Lifescience) according to manufacturer's instructions, or hsp65 DNA sequencing as previously described. Social-demographic data from patients were also analyzed and patients classified into 3 groups according only to microbiological data, "definite NTM disease", "NTM colonization" and, "possible NTM disease". RESULTS: In the period 2014-2020, the NRL-TB performed 50397 cultures. Among these, 1118 cultures were NTM positive retrieved from 944. Most of our cases were in patients whose mean age was 64±15.9 years, and no significant differences between gender was observed, although more frequent in male patients. Overall, from the 944 cases, we were able to identified 93 "definite NTM disease" cases and 79 "possible NTM disease". Mycobacterium avium complex (MAC) (40,8%), Mycobacterium abscessus-chelonae complex (MABC) (9,6%) and Mycobacterium fortuitum (6,3%) were responsible for most of the infections. The geographical distribution of NTM cases varied significantly and was possible to observe that was independent of population density. The region were most cases occurred was Lisbon Metropolitan Area (31,9%), followed by North (25,3%) and Centre (24,4%), however North region has the highest number of "definite NTM disease" cases (n=33). CONCLUSIONS: This is the first national wide epidemiological study on this subject, contributing to a better understanding of NTM dynamics in Portugal. MAC was the NTM species responsible for the majority of infections and, LMA the region with the highest number of cases. It was also possible to conclude that the number of NTM isolates is independent of the demography of the region.

Acute effect of passive cycle-ergometry and functional electrical stimulation on nitrosative stress and inflammatory cytokines in mechanically ventilated critically ill patients: a randomized controlled trial.

Early mobilization is beneficial for critically ill patients because it reduces muscle weakness acquired in intensive care units. The objective of this study was to assess the effect of functional electrical stimulation (FES) and passive cycle ergometry (PCE) on the nitrous stress and inflammatory cytometry in critically ill patients. This was a controlled, randomized, open clinical trial carried out in a 16-bed intensive care unit. The patients were randomized into four groups: Control group (n=10), did not undergo any therapeutic intervention during the study; PCE group (n=9), lower-limb PCE for 30 cycles/min for 20 min; FES group (n=9), electrical stimulation of quadriceps muscle for 20 min; and FES with PCE group (n=7), patients underwent PCE and FES, with their order determined randomly. The serum levels of nitric oxide, tumor necrosis factor alpha, interferon gamma, and interleukins 6 and 10 were analyzed before and after the intervention. There were no differences in clinical or demographic characteristics between the groups. The results revealed reduced nitric oxide concentrations one hour after using PCE (P<0.001) and FES (P<0.05), thereby indicating that these therapies may reduce cellular nitrosative stress when applied separately. Tumor necrosis factor alpha levels were reduced after the PCE intervention (P=0.049). PCE and FES reduced nitric oxide levels, demonstrating beneficial effects on the reduction of nitrosative stress. PCE was the only treatment that reduced the tumor necrosis factor alpha concentration.

Acute effect of passive cycle-ergometry and functional electrical stimulation on nitrosative stress and inflammatory cytokines in mechanically ventilated critically ill patients: a randomized controlled trial

Early mobilization is beneficial for critically ill patients because it reduces muscle weakness acquired in intensive care units. The objective of this study was to assess the effect of functional electrical stimulation (FES) and passive cycle ergometry (PCE) on the nitrous stress and inflammatory cytometry in critically ill patients. This was a controlled, randomized, open clinical trial carried out in a 16-bed intensive care unit. The patients were randomized into four groups: Control group (n=10), did not undergo any therapeutic intervention during the study; PCE group (n=9), lower-limb PCE for 30 cycles/min for 20 min; FES group (n=9), electrical stimulation of quadriceps muscle for 20 min; and FES with PCE group (n=7), patients underwent PCE and FES, with their order determined randomly. The serum levels of nitric oxide, tumor necrosis factor alpha, interferon gamma, and interleukins 6 and 10 were analyzed before and after the intervention. There were no differences in clinical or demographic characteristics between the groups. The results revealed reduced nitric oxide concentrations one hour after using PCE (P<0.001) and FES (P<0.05), thereby indicating that these therapies may reduce cellular nitrosative stress when applied separately. Tumor necrosis factor alpha levels were reduced after the PCE intervention (P=0.049). PCE and FES reduced nitric oxide levels, demonstrating beneficial effects on the reduction of nitrosative stress. PCE was the only treatment that reduced the tumor necrosis factor alpha concentration.

Insights into the population structure and pan-genome of Haemophilus influenzae.

The human-restricted bacterium Haemophilus influenzae is responsible for respiratory infections in both children and adults. While colonization begins in the upper airways, it can spread throughout the respiratory tract potentially leading to invasive infections. Although the spread of H. influenzae serotype b (Hib) has been prevented by vaccination, the emergence of infections by other serotypes as well as by non-typeable isolates (NTHi) have been observed, prompting the need for novel prevention strategies. Here, we aimed to study the population structure of H. influenzae and to get some insights into its pan-genome. We studied 305H. influenzae strains, enrolling 217 publicly available genomes, as well as 88 newly sequenced H. influenzae invasive strains isolated in Portugal, spanning a 24-year period. NTHi isolates presented a core-SNP-based genetic diversity about 10-fold higher than the one observed for Hib. The analysis of key factors involved in pathogenesis, such as lipooligosaccharides, hemagglutinating pili and High Molecular Weight-adhesins, suggests that NTHi shape its virulence repertoire, either by acquisition and loss of genes or by SNP-based diversification, likely towards host immune evasion and persistence. Discreet NTHi subpopulations structures are proposed based on core-genome supported with 17 candidate genetic markers identified in the accessory genome. Additionally, this study provides two bioinformatics tools for in silico rapid identification of H. influenzae serotypes and NTHi clades previously proposed, obviating laboratory-based demanding procedures. The present study constitutes an important genomic framework that could lay way for future studies on the genetic determinants underlying invasiveness and disease and population structure of H. influenzae.

Transplantation of Human Adipose Mesenchymal Stem Cells in Non-Immunosuppressed GRMD Dogs is a Safe Procedure.

The possibility to treat Duchenne muscular dystrophy (DMD), a lethal X-linked disorder, through cell therapy with mesenchymal stromal cells (MSCs) has been widely investigated in different animal models. However, some crucial questions need to be addressed before starting human therapeutic trials, particularly regarding its use for genetic disorders. How safe is the procedure? Are there any side effects following mesenchymal stem cell transplantation? To address these questions for DMD the best model is the golden retriever muscular dystrophy dog (GRMD), which is the closest model to the human condition displaying a much longer lifespan than other models. Here we report the follow-up of 5 GRMD dogs, which were repeatedly transplanted with human adipose-derived mesenchymal stromal cells (hASC), derived from different donors. Xenogeneic cell transplantation, which was done without immunosuppression, was well tolerated in all animals with no apparent long-term adverse effect. In the present study, we show that repeated heterologous stem-cell injection is a safe procedure, which is fundamental before starting human clinical trials.

Predição de modelos sobre a cinética de secagem de folhas de Ziziphus joazeiro Mart / Prediction models in the kinetics of drying leaves of Ziziphus joazeiro Mart

RESUMO: Esta pesquisa objetivou estudar, experimentalmente, a cinética de secagem de folhas de juazeiro (Ziziphus joazeiro Mart.) sob camada delgada em estufa com circulação forçada de ar utilizando temperaturas de 40, 50 e 60 ºC e velocidade do ar de 0,5, 1,0 e 1,5 m.s-1 conforme planejamento fatorial (22 + 3) e posterior ajuste das equações matemáticas aos dados experimentais, verificando assim aquele que melhor representa o fenômeno de secagem. Foi determinado o teor de água inicial das folhas utilizando o método padrão da estufa, em triplicatas. Para cada tratamento de secagem foram utilizados em torno de 150 g de folhas. Para o ajuste utilizou-se análise de regressão não linear, pelo método Quasi-Newton, por meio do programa computacional Statistica 5.0®, em que os valores dos parâmetros das equações foram estimados em função da temperatura e da velocidade do ar de secagem. A equação que melhor representou o processo de secagem do juazeiro para a faixa de temperatura de 40 a 60 ºC foi a de Midilli. Os resultados mostraram que com o acréscimo da temperatura ocorre maior redução no tempo de secagem.

ABSTRACT: This research aimed to study experimentally the kinetics of drying leaves of Ziziphus joazeiro Mart. in thin layer in an oven with forced air using the temperatures of 40, 50 and 60 ºC and the air velocity of 0.5, 1.0 and 1.5 m s-1 according to the factorial design (22 + 3) and subsequent adjustment of the experimental data in different models, and we select the one that best represents the phenomenon of drying. We determined the initial water content of the leaves by the standard oven method, in triplicates. For each drying treatment, we used approximately 150g of leaves. For adjustment, we used the non-linear regression analysis, through the Quasi-Newton method, using the software Statistica 5.0 ®, in which the parameter values of the equations were determined for the temperature and velocity of the drying air. The equation that best represented the drying process of the Ziziphus joazeiro Mart. for the temperature range of 40-60 ºC was the Midilli equation. The results showed that there is a greater reduction in drying time with increasing temperature.

Isolation and molecular characterization of Toxoplasma gondii isolated from pigeons and stray cats in Lisbon, Portugal.

Cats and pigeons are important factors in the epidemiology of Toxoplasma gondii as felids are the only definitive hosts that can excrete environmentally resistant oocysts, and pigeons share the same places of cats and humans constituting a good model and indicator of the ground field contamination. We aimed to study the virulence and genotypes of T. gondii isolated from pigeons and stray cats in Lisbon, Portugal. Fresh samples of brain from 41 pigeons and 164 cats revealing antibodies to T. gondii were inoculated in mice. Three isolates (one isolated from a cat and two isolated from pigeons) were virulent in the mouse model. Sag2-based genotyping of T. gondii was achieved in 70.7% (29/41) of samples isolated from pigeons (26 samples were type II, two were type III, and one strain was type I). From the cat brain samples, 50% (82/164) yielded Sag2 positive results, where 72 belonged to genotype II and 10 were no type III (it was not possible to discriminate between type I and II). Further genotyping was obtained by multiplex PCR of 5 microsatellites (TUB2, TgM-A, W35, B17, B18), allowing the identification of two recombinant strains that had been previously identified as type II by Sag2 amplification (one isolated from cat brain and the other from pigeon brain). This is the first evidence of recombinant strains circulating in Portugal and the first report of T. gondii genotyping from cats in this country. This study also highlights the importance of environmental contamination in the synanthropic cycle constituting a potential source of human infection.

Human adipose tissue derived pericytes increase life span in Utrn (tm1Ked) Dmd (mdx) /J mice.

Duchenne muscular dystrophy (DMD) is still an untreatable lethal X-linked disorder, which affects 1 in 3500 male births. It is caused by the absence of muscle dystrophin due to mutations in the dystrophin gene. The potential regenerative capacity as well as immune privileged properties of mesenchymal Stem Cells (MSC) has been under investigation for many years in an attempt to treat DMD. One of the questions to be addressed is whether stem cells from distinct sources have comparable clinical effects when injected in murine or canine muscular dystrophy animal models. Many studies comparing different stem cells from various sources were reported but these cells were obtained from different donors and thus with different genetic backgrounds. Here we investigated whether human pericytes obtained from 4 different tissues (muscle, adipose tissue, fallopian tube and endometrium) from the same donor have a similar clinical impact when injected in double mutant Utrn (tm1Ked) Dmd (mdx) /J mice, a clinically relevant model for DMD. After a weekly regimen of intraperitoneal injections of 10(6) cells per 8 weeks we evaluated the motor ability as well as the life span of the treated mice as compared to controls. Our experiment showed that only adipose tissue derived pericytes are able to increase significantly (39 days on average) the life span of affected mice. Microarray analysis showed an inhibition of the interferon pathway by adipose derived pericytes. Our results suggest that the clinical benefit associated with intraperitoneal injections of these adult stem cells is related to immune modulation rather than tissue regeneration.

Espécies bioativas em áreas úmidas do Planalto Catarinense / Bioactive species in wetlands of the uplands of Santa Catarina

O objetivo deste trabalho foi levantar e identificar as espécies vegetais que ocorrem em áreas úmidas (banhados) do Planalto Catarinense e associá-las aos seus respectivos potenciais bioativos, validados em testes laboratoriais ou relatados em estudos etnobotânicos. O estudo foi realizado em 12 áreas de banhados localizadas em fazendas com reflorestamentos comerciais de espécies exóticas de propriedade da Empresa Klabin S.A. A amostragem da vegetação foi realizada por meio de transecção no sentido transversal na maior largura dos banhados e na área de transição destes com a área mais drenada (em faixa limitada de três metros), onde foram coletados os espécimes férteis a cada metro da transecção. Após a identificação das espécies foi realizada uma revisão bibliográfica sobre o potencial bioativo das mesmas junto à bases de dados científicos utilizando-se os trabalhos que atestam o potencial e também os trabalhos que valorizam o conhecimento de populações locais. Nas áreas amostradas foram identificadas 235 espécies classificadas em 40 famílias botânicas. Destas, 28 espécies, classificadas em 11 famílias, possuem potencial bioativo. Do total de espécies identificadas, 18 apresentaram potencial validado cientificamente e 10 são citadas quanto ao seu potencial em trabalhos de etnobotânica. Ainda é pouco conhecida a diversidade de banhados no Planalto Catarinense sendo necessário mais estudo para o conhecimento da flora local. O estudo mostrou um grande número de espécies com potencial validado e que poderiam ser utilizadas pela população, enquanto outras que merecem pesquisas complementares.

Our objective was to survey and identify the species that occur in wetlands of the uplands of the state of Santa Catarina and associate them to their bioactive potential, validated by laboratory tests or reported in ethnobotanical studies. The study was conducted in 12 wetland areas, located in farms with commercial reforestation of exotic species (these areas belong to the Klabin S.A. Company). The sampling of the vegetation was performed by transection in the transverse direction, in the widest portion of the wetlands and in the transition area to the drier portion (a limited strip of approximately 3 meters). Fertile specimens were collected at every meter of the transection. After the identification of the species, a literature review on the bioactive potential of these species was carried out on the scientific databases, using the studies that attest the potential value of the species and also studies that value the expertise from local populations. In the sampled areas, 235 species were identified and classified into 40 botanical families. From these, 28 species, classified into 11 families, have bioactive potential. Among all species identified, 18 showed scientifically accredited bioactive potential and 10 were cited as to their potential in ethnobotanical studies. Little is known about the diversity of the wetlands in the uplands of the state of Santa Catarina, and further studies are needed to increase the knowledge on the local flora. This study showed a large number of species with validated potential and that could be used by the population, while others have not yet been studied, but that are potential candidates for further researches.

Selection of reference genes for real-time expression studies in Streptococcus agalactiae.

Streptococcus agalactiae, group B streptococci (GBS) is the leading cause of severe bacterial infections in newborns. GBS expression studies allowed the identification and characterization of virulence factors and a better understanding of the host-pathogen-environment interactions. The measurement of transcript levels by quantitative real-time PCR (qRT-PCR) is a widely used technique in GBS; however, a systematic evaluation and validation of reference gene stability for normalization purposes in GBS expression studies is currently lacking. Therefore, we analyzed the stability of 10 candidate reference genes (16SrRNA, glcK, glnA, groEL, gyrA, recA, rpoB, rpsL, sdhA and tkt) in three GBS prototype strains (O90R, NEM316 and 2603V/R) grown at different temperature conditions (37°C and 40°C). Our approach was based on the calibration of transcript levels from each gene against the number of bacteria from the same sample (ratio messenger RNA/genomic DNA). As a complementary analysis, reference gene stability was also investigated through the bioinformatic applications, geNorm and NormFinder. Considering the whole GBS development cycle, only a minority of genes were stable under both growth conditions, but this number increased when restricting the analysis to the logarithmic time-points. The range of stable genes was higher at 37°C, where recA and sdhA were stable simultaneously for the three strains, and six out of 10 genes were stable for at least two strains. At 40°C, recA showed up again as one of the best options, suggesting its potential use as reference gene in future qRT-PCR studies. The results generated with geNorm and NormFinder were consistent with those obtained experimentally and evidenced minor variations either among strains or temperature conditions. In conclusion, the fluctuation of expression of reference genes observed among different GBS strains and growth conditions highlights the importance of carefully validating, for each experimental scenario, the use of reference genes for qRT-PCR normalization purposes. Nevertheless, recA seems to be a good candidate for such optimizations.

Partner notification for chlamydia trachomatis urogenital infections: eight years of patient referral experience in the major Portuguese sexually transmitted infections clinic, 2000-07.

The aim of this study was to perform a retrospective evaluation of the success of patient referral for partner notification of chlamydial infections to the major Portuguese sexually transmitted infection (STI) clinic. A total of 8277 patients were screened for Chlamydia trachomatis during 2000-2007, and 695 (8.4%) tested positive. The sexual partners of 34% of these index cases attended the clinic as contacts following partner referral. In univariate analysis, heterosexual men referred partners more frequently than men who have sex with men (MSM) and HIV-negative index cases referred partners four-fold more frequently than HIV-positives; however, these associations were non-significant after multivariate analysis (adjusted odds ratio [OR] 1.4, 95% confidence interval [CI] 0.7-2.6; adjusted OR 4.2, 95% CI 0.9-18.7, respectively). Index patients who reported lower numbers of partners referred more frequently, and this association remained significant after multivariate analysis. One-third of the referred individuals tested C. trachomatis-positive; the risk for infection was three-fold higher for referred partners from symptomatic index cases (P < 0.001, adjusted OR, 95% CI 1.8-6.3). In conclusion, the results of the present study concerning sexual behaviour, HIV status and clinical signs highlight the need for further evaluations that may shape future partner notification strategies in order to reduce the chlamydial disease burden.

Normalization strategies for real-time expression data in Chlamydia trachomatis.

Chlamydia trachomatis is a widespread obligate intracellular pathogen genetically non-tractable for which transcriptomics is a fundamental tool to better understand its biology. However, the suitability of endogenous controls for normalization of transcriptomic data in this bacterium still needs validation. We aimed to assess the stability of 10 genes for their potential use as endogenous controls in real-time quantitative PCR assays at both normal and stress (D-cycloserine treatment) growth conditions throughout the developmental cycle of three C. trachomatis strains with different tissue tropism. Normalization was performed by real-time absolute quantification of the bacterial genomes. We also tested the applicability of two widely used softwares (geNorm and Normfinder) to our data. For all strains, we found that 16SrRNA was the most stably expressed gene throughout the chlamydial normal developmental cycle, which indicates its potential use as endogenous control in relative expression assays. However, it was highly unstable under D-cycloserine treatment (where oppA_2 was top-ranked), suggesting prudence when using ribosomal genes in expression experiments involving stress conditions. The geNorm and Normfinder algorithms revealed contrasting results and seem inappropriate for the selected pool of genes. Considering the multiplicity of experimental conditions, there should be an in loco validation of endogenous controls, where 16SrRNA appears to be in the front line. Alternatively, normalization of expression data against genomic DNA, which is less influenced by experimental constraints that are especially relevant for intracellular organisms, likely constitutes a good option. Moreover, the number of genomes also seems to be less subject to variation than expression of endogenous controls when working under stress conditions. The present study constitutes the first evaluation of putative endogenous controls for real-time expression assays in C. trachomatis.

Molecular characterization and antimicrobial susceptibility profiles in Streptococcus agalactiae colonizing strains: association of erythromycin resistance with subtype III-1 genetic clone family.

Knowledge of the epidemiology of Streptococcus agalactiae in Portugal is limited: therefore, the present study aimed to investigate the carriage rate of S. agalactiae among Portuguese women of reproductive age and the prevalence of antibiotic resistance, as well as to perform a molecular characterization of the clinical isolates. S. agalactiae was recovered from 6.2% of 4269 women during the period 2005­2007, with a predominance of capsular genotypes III (35%), V (33%), Ia (16%) and II (10%) in a sample of 100 isolates. To our knowledge, this is the first report of the S. agalactiae colonization rate in Portugal determined according to CDC guidelines. All isolates were susceptible to penicillin and vancomycin, whereas resistance to clindamycin and erythromycin was detected in 10% and 19% of isolates, respectively. Among the 19 erythromycin-resistant isolates, ten (53%) displayed the constitutive MLS(B) phenotype (conferring high-level resistance to macrolides), eight (42%) had the inducible MLS(B), and the M phenotype accounted for one isolate (5%). erm methylase genes were exclusively associated with MLS(B) phenotype isolates, whereas the M phenotype was a result of the presence of mefA. Multilocus sequence typing analysis of the genetic relatedness among isolates presenting resistance to erythromycin demonstrated a novel association between erythromycin resistance and the subtype III-1/ST-19 genetic clone family.

Molecular typing of treponema pallidum clinical strains from Lisbon, Portugal.

A molecular system was used to subtype Portuguese Treponema pallidum clinical strains isolated from both skin lesions and blood. The study with this system constitutes the first typing study in a European country. Three T. pallidum subtypes were found: subtypes 14a (50%), 14d (45.2%), and 14f (4.8%). Further studies are needed to better characterize the isolates involved in syphilis outbreaks.

Sensitivity evaluation of the Gen-Probe AMP-CT assay by pooling urine samples for the screening of Chlamydia trachomatis urogenital infection.

The sensitivity of two urine pool sizes versus individual testing, to detect Chlamydia trachomatis urogenital infection, was evaluated using the Gen-Probe AMP-CT assay. Thirty-three (33) known polymerase chain reaction (PCR) positive urine specimens were combined with 231 fresh first-catch urine (FCU) samples in 33 groups of four and 33 groups of eight, to make up 4X and 8X pooled samples, respectively. Gen-Probe AMP-CT assay was performed on pools as well as on individual samples at the same time. For the discrepant cases, the known positive samples were diluted 1:4 and 1:8 using the manufacturer's dilution buffer and were retested. Additional positive specimens found among fresh FCU samples were also tested by the Amplicor-PCR assay to confirm their positivity. The sensitivities of 8X pooling, 4X pooling and individual testing were 86.5%, 94.3% and 91.9%, respectively. The Gen-Probe AMP-CT assay applied to a 4X urine pooling model was highly sensitive and may be useful for a population based screening programme.

Neisseria gonorrhoeae and Chlamydia trachomatis infections in patients attending STD and family planning clinics in Bissau, Guinea-Bissau.

Accurate clinical and laboratory data about sexually transmitted diseases (STD) prevalence in Guinea-Bissau are not available. These data are important, since HIV2 is prevalent in this country, rates of HIV1 are increasing and STDs facilitate HIV transmission. Since DNA amplification methods have demonstrated to accurately diagnose chlamydial infections and gonorrhoea, the Amplicor CT/NG PCR Assay with Internal Control of Amplification (Roche Diagnostic System, Branchburg, NJ, USA) was used to estimate the prevalence of Neisseria gonorrhoeae and Chlamydia trachomatis genital infections in STDs and Family Planning Clinic attenders in Bissau, from March to July 1997. Two hundred and two cervical swabs and 31 urethral swabs were examined. Two women were excluded from this study because their cervical swabs contained inhibitory substances. N. gonorrhoeae was identified in 34/200 (17%) women and in 12/31 (38.7%) men. C. trachomatis was detected in 8/200 (4%) women there were no positive C. trachomatis results among the 31 men with urethritis. One woman presented a mixed infection with both organisms. The prevalence difference between men and women was not statistically significant (P=0.6) for C. trachomatis infection, but it was significant for N. gonorrhoeae infection (P=0.01). The prevalence rates of these infections found in this study, support the need for an urgent strategy to control STD in the region.

High-level tetracycline resistant Neisseria gonorrhoeae isolated in Portugal.

The first high-level tetracycline resistance (MIC > or = 16 mg/l) isolates of Neisseria gonorrhoeae (TRNG) were reported in 1990 from patients attending a Sexual Transmitted Disease (STD) Center in Lisbon. The TRNG prevalence was 4% in 1991, 5.3% in 1992 and 10,8% in 1994, exploding to 52.2% in 1995. The tet M determinant was evaluated by PCR. The digests of PCRP using HpaII produced the restriction pattern 2 for all the strains, except one (pattern 3). 78.3% of the TRNG strains were beta-lactamase producers and the 4.5 MDa penicillinase plasmid was the dominant (83%), 90% and 93.3% of the TRNG strains belonged to the auxotype NR and to the serogroup IA, respectively. The IA-8/NR class represented 58.3% of the TRNG isolates, suggesting a clonal spreading.