Hantaviruses are the causative agents of haemorrhagic fever with renal syndrome (HFRS) in Eurasia and of hantavirus cardiopulmonary syndrome (HCPS) in the Americas. The case fatality rate varies between different hantaviruses and can be up to 40%. At present, there is no specific treatment available. The hantavirus pathogenesis is not well understood, but most likely, both virus-mediated and host-mediated mechanisms are involved. The aim of the present study was to investigate the association among Puumala hantavirus (PUUV) viral RNA load, humoral immune response and disease severity in patients with HFRS. We performed a study of 105 PUUV-infected patients that were followed during the acute phase of disease and for up to 1-3 months later. Fifteen of the 105 patients (14%) were classified as having moderate/severe disease. A low PUUV-specific IgG response (p <0.05) and also a higher white blood cell count (p <0.001) were significantly associated with more severe disease. The PUUV RNA was detected in a majority of patient plasma samples up to 9 days after disease onset; however, PUUV RNA load or longevity of viraemia were not significantly associated with disease severity. We conclude that a low specific IgG response was associated with disease severity in patients with HFRS, whereas PUUV RNA load did not seem to affect the severity of HFRS. Our results raise the possibility of passive immunotherapy as a useful treatment for hantavirus-infected patients.

Sputum smear microscopy for the diagnosis of tuberculosis (TB) is cheap and simple but its sensitivity is low. Culture on Löwenstein-Jensen (LJ) is more sensitive but it takes a long time to yield results. Thin-Layer Agar (TLA) culture was suggested as an equally sensitive and faster alternative. We evaluated the performance of TLA for diagnosing TB in Jogjakarta, Indonesia. People with suspected TB presenting from July 2010 to July 2011 to two chest clinics of the National TB Control Programme network of Jogjakarta were eligible for inclusion. A sputum sample was sent to the Gadjah Mada University microbiology laboratory for concentration, smearing, Ziehl-Neelsen staining and culture on LJ and TLA. Sensitivity of cultures was evaluated against a composite reference standard (any positive culture). Time to detection of Mycobacteria was recorded. Out of 1414 samples, 164 (12%) were smear positive, 99 (7%) were scanty and 1151 (81%) were negative. On TLA and LJ respectively, 168 (12%) and 149 (11%) samples were positive, 72 (5%) and 32 (2%) were contaminated (κ = 0.64; 95% CI 0.59-0.69, p <0.01). Using the reference standard, 196 (14%) TB cases were identified. The sensitivity of TLA was 0.86 (95% CI 0.80-0.90), significantly higher (p 0.03) than for LJ (0.76; 95% CI 0.69-0.81). The median time to detection in days was significantly shorter (p <0.01) for TLA (12; 95% CI 11-13) than for LJ (44; 95% CI 43-45). TLA is a rapid and sensitive method for the diagnosis of TB. Implementation studies to evaluate the cost-effectiveness and impact of its introduction into programmatic settings are urgently needed.

We investigated whether dried cerebrospinal fluid (CSF) conserved on filter paper can be used as a substrate for accurate PCR diagnosis of important causes of bacterial meningitis in the Lao PDR. Using mock CSF, we investigated and optimized filter paper varieties, paper punch sizes, elution volumes and quantities of DNA template to achieve sensitive and reliable detection of bacterial DNA from filter paper specimens. FTA Elute Micro Card™ (Whatman, Maidstone, UK) was the most sensitive, consistent and practical variety of filter paper. Following optimization, the lower limit of detection for Streptococcus pneumoniae from dried mock CSF spots was 14 genomic equivalents (GE)/μL (interquartile range 5.5 GE/μL) or 230 (IQR 65) colony forming units/mL. A prospective clinical evaluation for S. pneumoniae, S. suis and Neisseria meningitidis was performed. Culture and PCR performed on fresh liquid CSF from patients admitted with a clinical diagnosis of meningitis (n = 73) were compared with results derived from dried CSF spots. Four of five fresh PCR-positive CSF samples also tested PCR positive from dried CSF spots, with one patient under the limit of detection. In a retrospective study of S. pneumoniae samples (n = 20), the median (IQR; range) CSF S. pneumoniae bacterial load was 1.1 × 10(4) GE/μL (1.2 × 10(5) ; 1 to 6.1 × 10(6) DNA GE/μL). Utilizing the optimized methodology, we estimate an extrapolated sensitivity of 90%, based on the range of CSF genome counts found in Laos. Dried CSF filter paper spots could potentially help us to better understand the epidemiology of bacterial meningitis in resource-poor settings and guide empirical treatments and vaccination policies.

The pork-processing industry has been possibly the fastest growing sector of the food industry in recent years. Specialization, genetic homogenization of the pig population, high density of the breeding population, reduced human-animal interactions, slaughter at a lower age and increased international trade of live animals and pork are parameters that affect, positively or negatively, the emergence of novel pig-borne pathogens, many of which are pig-specific, and many of which have significant zoonotic potential, as observed in recent outbreaks of Nipah virus and Streptococcus suis in Southeast Asia and China, respectively. Numerous other pathogens are transmitted to humans through direct contact with or consumption of pig products, and globalization trends in trade and human population movements have resulted in outbreaks of pig-borne diseases even in Muslim countries and in Israel, where pork consumption is religiously prohibited. The role of pigs as potential reservoirs of antibiotic-resistant pathogens or genes encoding resistance, and the role of feral pigs as a reservoir of zoonotic disease, are scientific fields in direct need of further research.

Tuberculosis (TB) has re-emerged over the past two decades: in industrialized countries in association with immigration, and in Africa owing to the human immunodeficiency virus epidemic. Drug-resistant TB is a major threat worldwide. The variable and uncertain impact of TB control necessitates not only better tools (diagnostics, drugs, and vaccines), but also better insights into the natural history and epidemiology of TB. Molecular epidemiological studies over the last two decades have contributed to such insights by answering long-standing questions, such as the proportion of cases attributable to recent transmission, risk factors for recent transmission, the occurrence of multiple Mycobacterium tuberculosis infection, and the proportion of recurrent TB cases attributable to re-infection. M. tuberculosis lineages have been identified and shown to be associated with geographical origin. The Beijing genotype is strongly associated with multidrug resistance, and may have escaped from bacille Calmette-Guérin-induced immunity. DNA fingerprinting has quantified the importance of institutional transmission and laboratory cross-contamination, and has helped to focus contact investigations. Questions to be answered in the near future with whole genome sequencing include identification of chains of transmission within clusters of patients, more precise quantification of mixed infection, and transmission probabilities and rates of progression from infection to disease of various M. tuberculosis lineages, as well as possible variations in vaccine efficacy by lineage. Perhaps most importantly, dynamics in the population structure of M. tuberculosis in response to control measures in high-prevalence areas should be better understood.

Human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) are major public health concerns. We aimed to determine the prevalence of HBV and HCV infections among HIV-infected patients, and to identify the main circulating hepatitis strains in Morocco. The study was carried out in 503 HIV-infected patients. Our survey indicated that the prevalence of HIV/hepatitis co-infection was 10.6%; 5.2% of patients were HBV surface antigen positive, and 5.4% of patients were anti-HCV positive. Among the HBV surface antigen-positive group, HBV DNA sequencing identified exclusively genotype D (D1: 26.7%; D7: 73.3%) in accordance with what is found in the general population. In contrast, sequencing of HCV isolates produced an unusual subtype distribution with a decreasing order of prevalence: 1a, 3a (both 23.5%), 1b, 4a (both 17.6%), 1c (11.8%) and 6h (6%).

The lymphogranuloma venereum (LGV) outbreak described in the Netherlands in 2003, increased the interest in the genotyping of Chlamydia trachomatis. Although international surveillance programmes were implemented, these studies slowly decreased in the following years. Now data have revealed a new accumulation of LGV cases in those European countries with extended surveillance programmes. Between March 2009 and November 2011, a study was carried out to detect LGV cases in Madrid. The study was based on screening of C. trachomatis using commercial kits, followed by real-time pmpH-PCR discriminating LGV strains, and finally ompA gene was sequenced for phylogenetic reconstruction. Ninety-four LGV infections were identified. The number of cases increased from 10 to 30 and then to 54 during 2009-2011. Incidence of LGV was strongly associated with men who have sex with men; but in 2011, LGV cases were described in women and heterosexual men. Sixty-nine patients were also human immunodeficiency virus (HIV) positive, with detectable viral loads at the moment of LGV diagnosis, suggesting a high-risk of co-transmission. In fact, in four patients the diagnosis of HIV was simultaneous with LGV infection. The conventional treatment with doxycycline was prescribed in 75 patients, although in three patients the treatment failed. The sequencing of the ompA gene permitted identification of two independent transmission nodes. One constituted by 25 sequences identical to the L2b variant, and a second node including 37 sequences identical to L2. This epidemiological situation characterized by the co-circulation of two LGV variants has not been previously described, reinforcing the need for screening and genotyping of LGV strains.

A rapid and sensitive H7 and N9 subtype-specific reverse transcription loop-mediated isothermal amplification assay was developed respectively for visual detection of human-infected influenza A (H7N9) virus. The reaction was performed in one step in a single tube at 63°C for 60 min with the addition of hydroxynaphthol blue dye before amplification. The detection limits of both subtype-specific assays were comparable to those of validated H7 and N9 real-time PCR assays respectively and no cross-detection was observed with influenza A pandemic H1N1, H3N2, H5N1, H9N2 or influenza B virus. The assays were evaluated further with H7N9 virus-infected clinical specimens.