Absence of hybridization with the wild-type and mutant rpoB probes in the Genotype MTBDRplus assay detects 'disputed' rifampicin mutations.

OBJECTIVE: Mycobacterium tuberculosis isolates that fail to hybridize to at least one rpoB wild-type or any mutation probe on the Genotype MTBDRplus strip are assumed to be rifampicin-resistant. However, the precise mutation(s) are unknown. We sought to identify the mutations in isolates with such hybridization patterns and determine if the mutations are associated with resistance to rifampicin. METHODS: In this study, 275 M. tuberculosis isolates were screened with the Genotype MTBDRplus assay to identify isolates with the hybridization pattern. These isolates were sequenced and their minimum inhibitory concentrations (MIC) determined using the Bactec MGIT 960 system. RESULTS: Among the 275 isolates tested, 15 (6%) isolates with the hybridization pattern were identified. Sequencing showed that failure to hybridize to rpoB wild-type probes resulted from the presence of 'disputed' rifampicin mutations, which are mutations not always associated with a rifampicin-resistant phenotype. All, except 3/15, isolates had a rifampicin-resistant phenotype (MIC > 1 µg/mL). One of the three isolates with a rifampicin-susceptible phenotype had the same mutation at position 526 (His526Leu) as another isolate that had a rifampicin-resistant phenotype. CONCLUSION: The recommendation of the Genotype MTBDRplus assay to assume rifampicin resistance based solely on failure to hybridize to rpoB wild-type probe allows the identification of important RIF-resistant isolates. About 20% (3/15) of such isolates could be missed by relying only on the standard MGIT 960 DST assay for drug susceptibility testing.

Stepwise implementation of a new diagnostic algorithm for multidrug-resistant tuberculosis in Haiti.

SETTING: The uptake of tests endorsed by the World Health Organization to detect and appropriately confirm multidrug-resistant tuberculosis (MDR-TB) in low-income countries remains insufficient. OBJECTIVE: To validate the implementation of line-probe assays (LPA) and liquid culture to develop an algorithm to detect MDR-TB in the challenging setting of Haiti. METHODS: Through an EXPAND-TB (Expanding Access to New Diagnostics for TB) partnership, proficiency testing and validation of 221 acid-fast bacilli positive specimens were performed. Sensitivity, cost and processing time were analysed. RESULTS: Using liquid vs. solid culture shortened the turnaround time from 54 to 19 days, with a sensitivity of 100% vs. 98.6% and a total cost reduction of 13%. LPA detected all TB and MDR-TB cases at a lower cost than culture, in a mean time of 7.5 days. CONCLUSION: The combined use of molecular and liquid culture techniques accelerates the accurate diagnosis of TB and susceptibility testing against first-line drugs in a significantly shorter time, and is less expensive. The implementation of this new algorithm could significantly and accurately improve the screening and treatment follow-up of patients affected with TB and MDR-TB.

Detection of three human adenovirus species in adults with acute respiratory infection in China.

Human adenoviruses (HAdVs) are recognized as causal agents in a wide range of human diseases. However, researchers lack sufficient data on the exact HAdV species and serotypes associated with adult acute respiratory tract infections (ARTIs). To detect and characterize HAdV infections in adults in China, clinical specimens were collected from 10,310 adults with ARTIs from May 2005 to July 2010. The partial HAdV hexon gene was amplified by polymerase chain reaction (PCR), sequenced, and phylogenetically analyzed. HAdVs were detected in 86 samples (0.8%), of which 67 (77.9%) were species B (HAdV-3, -7, -11, and -14), 7 (8.1%) were species C (HAdV-1, -2, and -6), and 12 (14%) were species E (HAdV-4). HAdV-3 was the most frequently detected serotype (41/86, 47.7%), followed by HAdV-7 (13/86, 15.1%), HAdV-4 (12/86, 14.0%), and HAdV-11 (11/86, 12.8%). Patients 14-25 years old (60.5%) exhibited a higher rate of adenovirus detection than older patients. Co-infections with other respiratory viruses were observed in samples positive for HAdV species B and E. Human rhinovirus was the most commonly found virus in patients with HAdV infection. These findings provide baseline data for the surveillance and control of HAdV infection in China.

Laboratory-based diagnosis of pneumococcal pneumonia: state of the art and unmet needs.

In view of the increasing use of pneumococcal vaccines, especially in the developing world, there is a need for appropriate diagnostics to understand the aetiology of pneumonia, to define the burden of pneumococcal disease, and to monitor vaccine efficacy and effectiveness. This article summarizes a meeting on the diagnosis, detection and serotyping of pneumococcal disease organized by PATH and Fondation Mérieux (18-20 October 2009, Fondation Mérieux Conference Centre, Les Pensières, France). Workers and experts met to discuss the gaps in the microbiology-based diagnosis of Streptococcus pneumoniae disease, with special emphasis on pneumonia. The meeting was designed to evaluate the state of the art of pneumococcal diagnostics and serotyping methodologies, identify research and development needs, and propose new guidelines to public health authorities to support the introduction of vaccines. Regarding detection, the main recommendations were to encourage chest X-rays and antigen detection in urine. Large-scale studies are needed to evaluate the diagnostic utility of test algorithms that associate chest X-rays, antigen detection in urine, S. pneumoniae quantitative PCR in nasopharyngeal aspirates and sputum, and C-reactive protein or procalcitonin measurement in blood. Efforts should be focused on proteomics to identify pneumococcus-specific antigens in urine or host markers in blood expressed during pneumonia. It was recommended to develop S. pneumoniae typing capacities, to understand the epidemiology of pneumococcal disease, and to evaluate vaccine effectiveness. Simple and effective approaches are encouraged, and new technologies based on beads, microarrays or deep sequencing should be developed to determine, in a single test capsular serotype, resistance profile and genotype.

Detection of human bocavirus 3 in China.

Since its first identification in 2005, four species of human bocavirus (HBoV1-4) have been documented. HBoV1 and HBoV2 have been shown to be associated with respiratory tract illnesses, as well as with acute gastroenteritis (AGE), worldwide. However, reports on the prevalence, clinical significance, and molecular characteristics of the two most newly identified HBoV species, HBoV3 and HBoV4, are very limited. To detect and characterize HBoV3 and HBoV4 infections in children with AGE in China, stool specimens were collected from 366 children with AGE. HBoVs in these samples were amplified by nested polymerase chain reaction (PCR), sequenced, and phylogenetically analyzed. HBoVs were detected in 44 samples (12%), of which nine were HBoV1, 33 were HBoV2, and two were HBoV3. HBoV4 was not detected. Most HBoV-positive samples (35/44) were co-detected with other viral pathogens. Both HBoV3 samples were co-detected with rotavirus. Analysis of the HBoV3 (46-BJ07) genome sequence indicates that HBoV3 may be a recombinant derived from HBoV1 and HBoV2 or from HBoV1 and HBoV4. To our knowledge, this is the first report of HBoV3 in China. However, it is unclear whether HBoV3 is associated with AGE because of its low detection rate in AGE patients and its co-infection with other AGE-causing viruses.

Afriflu--international conference on influenza disease burden in Africa, 1-2 June 2010, Marrakech, Morocco.

The burden of influenza disease is to a large extent unknown for the African continent. Moreover, the interaction of influenza with common infectious diseases in Africa remains poorly described. Solid scientific evidence on the influenza disease burden in Africa is critical for the development of effective influenza vaccine policies. On 1st and 2nd June 2010 in Marrakech, Morocco, over eighty surveillance and influenza experts from 22 African countries as well as Europe and America met at the 'Afriflu' conference to discuss influenza challenges and solutions for the continent. During the meeting, participants exchanged their experiences and discussed a wide variety of topics related to influenza in Africa, including diagnosis, surveillance, epidemiology, and interventions. The meeting concluded with a pledge to improve influenza knowledge and awareness in Africa, with an emphasis on accurate determination of disease burden to help orient public health policies.

Evaluation of real-time nucleic acid sequence-based amplification for detection of Chikungunya virus in clinical samples.

The Chikungunya virus (CHIKV) is a member of the genus Alphavirus that is transmitted to humans by Aedes mosquitoes. In 2005 and 2006, the Indian Ocean island of La Réunion was hit with an unprecedented CHIKV fever outbreak that infected 300 000 people. In the present study, we describe the evaluation of real-time nucleic acid sequence-based amplification (RT-NASBA) for the detection of CHIKV in clinical samples. A co-extracted and co-amplified chimerical CHIKV RNA sequence was used as an internal control to eliminate false-negative results. The detection threshold of the assay was determined from quantified CHIKV-positive plasma, and estimated to be 200 copies per NASBA reaction. The specificity of the assay was determined using blast analyses and non-cross-reactivity using an O'nyong-nyong virus culture and 250 CHIKV RT-PCR-negative plasma samples. A 100 % specificity was found and no invalid result was obtained, showing the good quality of the nucleic acid extraction. The assay was then evaluated using 252 CHIKV-positive RT-PCR plasma samples. The samples were all tested positive, including those with low viral load. This evaluation showed that the RT-NASBA is a rapid (5 h from sample nucleic acid extraction to detection), sensitive, specific and reliable method for the routine diagnosis of CHIKV in clinical samples.

Prevalence of human respiratory viruses in adults with acute respiratory tract infections in Beijing, 2005-2007.

To determine the aetiological role and epidemiological profile of common respiratory viruses in adults with acute respiratory tract infections (ARTIs), a 2-year study was conducted in Beijing, China, from May 2005 to July 2007. Nose and throat swab samples from 5808 ARTI patients were analysed by PCR methods for common respiratory viruses, including influenza viruses (IFVs) A, B, and C, parainfluenza viruses (PIVs) 1-4, enteroviruses (EVs), human rhinoviruses (HRVs), respiratory syncytial virus (RSV), human metapneumovirus (HMPV), human coronaviruses (HCoVs) OC43, 229E, NL63, and HKU1, and adenoviruses (ADVs). Viral pathogens were detected in 34.6% of patient samples, and 1.6% of the patients tested positive for more than one virus. IFVs (19.3%) were the dominant agents detected, followed by HRVs (6.5%), PIVs (4.3%), EVs (3.2%), and HCoVs (1.1%). ADVs, RSV and HMPV were also detected (<1%). The viral detection rates differed significantly between infections of the lower and upper respiratory tracts in the sample population: PIVs, the second most commonly detected viral agents in lower acute respiratory tract infections (LRTIs), were more prevalent than in upper acute respiratory tract infections, indicating that the pathogenic role of PIVs in LRTIs should be investigated. Currently, this study is the largest-scale investigation of respiratory virus infections in China with multiple agent detection, providing baseline data for further studies of respiratory virus infections in adults with ARTIs.

European multicenter evaluation of high-density DNA probe arrays for detection of hepatitis B virus resistance mutations and identification of genotypes.

Polymorphisms along the hepatitis B virus (HBV) genome have an impact on disease outcome, sensitivity to antiviral treatment, escape from vaccination, and laboratory diagnosis. We have designed a diagnostic tool based on duplex amplification of the whole HBV genome and a high-density DNA chip designed to detect 245 mutations, 20 deletions, and 2 insertions at 151 positions and to determine the genotype of the virus in serum. Assay performances were evaluated with 170 samples, characterized by determination of viral load and sequencing of the Pol, S, and precore genes and the basal core promoter. One hundred fifty-three samples (90%) could be amplified and analyzed by the chip. Only two samples with more than 10(3) genome copies/ml could not be analyzed. Genotype had no impact on analytical sensitivity. Reproducibility studies showed no difference between repeats for codon and genotype determination. Genotype determination by sequencing and the chip were concordant in 148 of 151 samples. Twelve thousand one hundred sixty-one codons were analyzed by both techniques. Only 89.4% could be determined by sequencing, and among the remaining 11,335 codons, 92.8% were identical by sequencing and the chip. Failures to identify an amino acid by the chip were mainly due to reduced hybridization efficiency attributed to unexpected polymorphisms. Optimization of the chip-based reagent for the analysis of the HBV genome is ongoing. This first evaluation showed that DNA chip technology can provide important information in relation to the clinical management of chronic hepatitis B.

Immunology-related perturbations induced by copper and chitosan in carp (Cyprinus carpio L.).

Copper is used in treatment mixtures to control fungal diseases in vineyards. Its concentrations are relatively high in some aquatic ecosystems, and the main problem observed in this study was the antioxidant stress induced by this heavy metal. Copper toxicologic effects in aquatic organisms have prompted the demand for alternative use of low-toxicity molecules in culture treatments. Chitosan is a polymer with antifungal property similar to copper and may be an interesting biopesticide. Thus, it is necessary to investigate the potential toxicity of chitosan for aquatic animal health, either alone or in conjunction with copper. In this study, carp were exposed to two sublethal chitosan concentrations (75 and 150 mg/L) or to two sublethal copper concentrations (0.1 and 0.25 mg/L) or to a mixture of chitosan plus copper (75 mg/L and 0.1 mg/L, respectively). The results of the present study show that exposure to copper at environmentally realistic levels or to chitosan at sublethal concentrations may significantly stimulate various aspects of immune functions in carp such as nonspecific cellular immunity, represented by total immunoglobulin level, ceruloplasmin activity, and oxidative activity of phagocytes. This acute-phase inflammatory response induced separately by the two treatments was not observed, especially on phagocyte oxidative activity, when carp were exposed to the copper-chitosan mixture. This fact could be explained by a possible chelation of copper by chitosan decreasing the biodisponibility of the two products for immune cells. Thus, the immunotoxicologic impact of copper and chitosan on fish immune response would be less pronounced with the combined treatments than with separate treatments in an aquatic environment.

Detection of human immunodeficiency virus type 1 antiretroviral resistance mutations by high-density DNA probe arrays.

Genotypic resistance testing has become an important tool in the clinical management of patients infected with human immunodeficiency virus type 1 (HIV-1). Standard sequencing methodology and hybridization-based technology are the two principal methods used for HIV-1 genotyping. This report describes an evaluation of a new hybridization-based HIV-1 genotypic test of 99 clinical samples from patients infected mostly with HIV-1 subtype B and receiving treatment. This test combines RNA extraction with magnetic silica particles, amplification by nested reverse transcriptase PCR, and detection with high-density probe arrays designed to detect 204 antiretroviral resistance mutations simultaneously in Gag cleavage sites, protease, reverse transcriptase, integrase, and gp41. The nested reverse transcriptase PCR success rates at viral loads exceeding 1,000 copies/ml were 98% for the 2.1-kb amplicon that covers the Gag cleavage sites and the protease and reverse transcriptase genes, 92% for the gp41 amplicon, and 100% for the integrase amplicon. We analyzed 4,465 relevant codons with the HIV-1 DNA chip genotyping assay and the classic sequence-based method. Key resistance mutations in protease and reverse transcriptase were identified correctly 95 and 92% of the time, respectively. This test should be a valuable alternative to the standard sequence-based system for HIV-1 drug resistance monitoring and a useful diagnostic tool for simultaneous multiple genetic analyses.

Diagnosis of zoonotic viral encephalitis.

Human infections by zoonotic encephalitis viruses are usually asymptomatic or symptoms are not specific to these viruses. Some of them have high mortality and morbidity rates and most often no specific treatment exist. This emphasizes the need for a precise identification of arboviruses in clinical specimens from humans and animals. Because these diseases are frequent in developing countries and tend to emerge or re-emerge in others, diagnostic tools must detect the broadest possible range of viruses with a high sensitivity and this is a key factor for surveillance, control of transmission and prevention through vaccination. In countries with limited diagnostic infrastructures, low-cost and easy-to-use tests are required. The diagnosis of arboviral encephalitis has been significantly improved in the recent years. Sensitive ELISA assay to detect antibodies against many arboviruses in serum or CSF are commercially available and can be used to detect early infections. Immunochromatographic rapid tests for the detection of specific IgM that could be used on fingertip blood would be valuable tools in developing countries. A limitation of these serologic assays is their lack of specificity as many arboviruses are antigenically related. Virus isolation or molecular assays from different human or animal tissues are also important diagnostic tools. Molecular assays have been extensively described in the recent years. They are very sensitive and have the advantage over cell culture that specimen transportation is less critical. Real-time detection has even improved sensitivity and reduced time-to-result. Although the utility of molecular assays for the detection of arboviruses in mosquito pools has been demonstrated, an extensive validation of their pertinence in clinical settings is still required. The use of DNA-microarrays may further extend the range of viruses that can be detected in a single test and allow isolates typing for epidemiology purposes.

Metallothionein induction in aquatic oligochaete tubifex tubifex exposed to herbicide isoproturon.

Metallothioneins (MTs) are low-molecular-weight proteins mainly involved in metal ion detoxification. Recently it has been demonstrated that MTs participate in several cellular functions such as regulation of growth and antioxidative defenses. Moreover, pesticides can induce their synthesis. The aim of the current work was to determine the effects of isoproturon, either pure or formulated as Matin (suspension containing an isoproturon concentration of 500 g. L(-1)), on the metallothionein and total protein contents of the aquatic worm Tubifex tubifex. MT levels in exposed worms increased significantly after 7 and 15 days of exposure to a concentration of the herbicide of 50 mg. L(-1). Isoproturon reduced the metal (Cu, Zn, and Cd) content of metallothioneins, and it also increased the total protein content of the worms. These results suggest that MT induction may not be considered a specific biomarker of metal exposure but that it can be used as a nonspecific biomarker of the effect of isoproturon effect in aquatic worms.

Chitosan improves development, and protects Vitis vinifera L. against Botrytis cinerea.

We evaluated the potential of chitosan both to stimulate plant development and to induce protection from Botrytis cinerea in Vitis vinifera L. plantlets. The presence of 1.75% (v/v) chitogel in the culture medium was the optimal concentration for in vitro grapevine plantlet growth, as determined by measurements on enhancement of root and shoot biomass. Photosynthesis and related parameters were also stimulated in chitogel-treated plantlets. Chitogel reduced the development of Botrytis cinerea and induced cytological alterations to the pathogen. When challenged with the fungus, a significant decrease in disease incidence was observed in plants growing on medium supplemented with chitogel. Furthermore, exogenous foliar applications of chitogel to plantlets growing on chitogel-free medium sensitized them so as to be protected against Botrytis cinerea attack. Our results indicate that chitogel can be used in the vineyard as a means to attain protection against Botrytis cinerea and that its application may counteract the wide use of chemical pesticides.

Stimulation of antioxidant enzymes levels in carp (Cyprinus carpio L.) infected by Ptychobothrium sp. (Cestoda).

Increased antioxidant enzymatic activities were observed in carp parasitised by Ptychobothrium sp. when compared with healthy fish. This antioxidant response could contribute to neutralise the oxidative stress normally induced by parasitism.

Metallothionein induction in the aquatic oligochaete Tubifex tubifex exposed to the herbicide isoproturon.

Metallothioneins (MTs), are low molecular weight proteins, mainly involved in metal ion detoxification. Recently it has been demonstrated that MTs participate in several cellular functions such as regulation of growth, and anti-oxidative defenses. Moreover, pesticides can induce their synthesis. The aim of the current work was to determine the effects of isoproturon either pure or formulated as Matin (suspension containing 500 g x l(-1) isoproturon) on the metallothionein and total protein content of the aquatic worm Tubifex tubifex. MT levels in exposed worms increased significantly after 7 and 15 days of exposure to 50 mg x l(-1) of herbicide. Isoproturon reduced metal (Cu, Zn, and Cd) content of metallothioneins, and it also increased total protein content of the worms. The results suggest that MT induction may not be considered as a specific biomarker of metal exposure but it can be used as a non specific biomarker of isoproturon effect in the worm.

Oxyfluorfen toxic effect on S. obliquus evaluated by different photosynthetic and enzymatic biomarkers.

The effect of oxyfluorfen was investigated when alga Scenedesmus obliquus has been exposed to different concentrations (7.5, 15, and 22.5 microg x L(-1)) at 12, 24, and 48 hours of exposure. Toxicity test was done by using 13 biomarkers concerning growth rate, chlorophyll content and indicators of photosynthetic and antioxidant enzyme activities. The change of the 13 parameters showed a great variation of sensitivity indicating differences in parameters' suitability to be used as biomarkers when alga culture was exposed to oxyfluorfen toxicity. The order of sensitivity between those biomarkers was: Antenna size (ABS/RC) > Chlorophyll content > Catalase (CAT) > Operational PSII quantum yield (phiS(PSII)) > Glutathione S-transferase (GST) > Functional plastoquinone pool (Q(PQ)) > Glutathione reductase (GR) > Growth rate > Nonphotochemical quenching (QN) > Proton gradient quenching (Q(Emax)) > Ascorbate peroxidase (APX) > Photochemical quenching (Q(p)) > Maximum PSII quantum yield (Phi(PSII)). The effect of oxyfluorfen on the changes of those parameters was interpreted as a result of herbicide mode of action at molecular level of alga cellular system. This study indicated for some photosynthetic and enzymatic biomarkers to be useful indicators of toxicity effect induced in non-target alga species. Determination of biomarkers' sensitivity order may facilitate their selection to be used in environmental risk assessment of polluted water.

Photosynthetic responses of Lemna minor exposed to xenobiotics, copper, and their combinations.

The effects on the photosynthetic process of copper and pesticides, used in vineyards, and their combinations, were investigated by measuring different chlorophyll fluorescence parameters in Lemna minor. Cu and flumioxazin had a severe impact on duckweed since a decrease in their photosynthetic capacity was detected after 24h of exposure to 200 and 1 microg.L(-1), respectively. However, fungicides used to control Botrytis cinerea (procymidone, pyrimethanil, and fludioxonil) seem to have no marked effects on duckweed even at very high concentrations (50 mg.L(-1)). Analysis of the combinations between copper (200 microg.L(-1)) and pesticides revealed different patterns of response: a synergistic effect was observed when Cu(2+) was added to flumioxazin (1 microg.L(-1)). In contrast, an antagonism was detected when duckweed was exposed to a mixture of Cu(2+) and fludioxonil or procymidone. However, these interactions always tended toward additivity when pesticide concentrations increased. Additivity was also observed for the Cu(2+)-pyrimethanil mixture at each fungicide concentration.

Antioxidant response modulated by copper in healthy or parasitized carp (Cyprinus carpio L.) by Ptychobothrium sp. (Cestoda).

An increased antioxidant response (catalase, glutathione S-transferase (GST) and glutathione reductase (GRd) activities in liver and GST activity in head kidney) was observed in carp parasitized by Ptychobothrium sp. compared to healthy fish. In case of a copper contamination of these fish, the decrease in enzymatic activities observed was less pronounced in parasitized than in healthy carp.