Genetic diversity of mycobacterium tuberculosis in northern Greece.

The aim of the present study was to describe the genetic diversity of Mycobacterium tuberculosis (M.tuberculosis) strains circulating in the region of Northern Greece. A total of thirty-seven M. tuberculosis clinical isolates were analysed by the spoligotyping method. According to the results, six clusters comprising seventeen strains were detected, and the remaining twenty strains showed unique patterns. The M.tuberculosis families according to SITVITWEB were distributed as follows: Haarlem (H) (27.0%); T (24.3%); Beijing (13.5%); Latin-America and Mediterranean (LAM) (5.4%) and S (2.7%). The remaining isolates (27%) did not match any isolates within the database and they were characterized as orphans. Regarding GenoType MTBDRplus results, two strains (5.4%) were Multi-Drug-Resistant, four strains (10.8%), were isoniazid monoresistant, while the remaining thirty-one strains (83.8%) were susceptible. In conclusion, in the region of Macedonia-Thrace (Northern Greece), there was high phylogenetic diversity among M. tuberculosis isolates. Molecular tools used and data presented can have regional and national impact on tuberculosis control.

Whole genome sequencing of Mycobacterium tuberculosis for detection of drug resistance: a systematic review.

OBJECTIVES: We conducted a systematic review to determine the diagnostic accuracy of whole genome sequencing (WGS) of Mycobacterium tuberculosis for the detection of resistance to first- and second-line anti-tuberculosis (TB) drugs. METHODS: The study was conducted according to the criteria of the Preferred Reporting Items for Systematic Reviews group. A total of 20 publications were included. The sensitivity, specificity, positive-predictive value and negative-predictive value of WGS using phenotypic drug susceptibility testing methods as a reference standard were determined. RESULTS: Anti-TB agents tested included all first-line drugs, a variety of reserve drugs, as well as new drugs. Polymorphisms in a total of 53 genes were tested for associations with drug resistance. Pooled sensitivity and specificity values for detection of resistance to selected first-line drugs were 0.98 (95% CI 0.93-0.98) and 0.98 (95% CI 0.98-1.00) for rifampicin and 0.97 (95% CI 0.94-0.99) and 0.93 (95% CI 0.91-0.96) for isoniazid, respectively. Due to high heterogeneity in study designs, lack of data, knowledge of resistance mechanisms and clarity on exclusion of phylogenetic markers, there was a significant variation in analytical performance of WGS for the remaining first-line, reserved drugs and new drugs. CONCLUSIONS: Whole genome sequencing could be considered a promising alternative to existing phenotypic and molecular drug susceptibility testing methods for rifampicin and isoniazid pending standardization of analytical pipelines. To ensure clinical relevance of WGS for detection of M. tuberculosis complex drug resistance, future studies should include information on clinical outcomes.

Pilot study of the completeness of notification of adult tuberculosis in Athens, Greece.

SETTING: Sotiria Chest Diseases Hospital (SCDH), a referral hospital in Athens, Greece, 2012. OBJECTIVE: To assess the completeness of the mandatory notification system for tuberculosis (TB) at the SCDH, and compare the observed and estimated annual incidence rates. DESIGN: Record linkage and the capture-recapture method were applied. Data sources were the registers from the national mandatory notification register (Hellenic Centre for Disease Control and Prevention [HCDCP]), the National Reference Laboratory for Mycobacteria (NRLM) and SCDH records. The log-linear model with the lowest Akaike information criterion was selected as the most valid statistical model. RESULTS: The observed and estimated TB under-reporting rates at the national level were respectively 55% (95%CI 49-60) and 75% (95%CI 71-78). The observed completeness of the HCDCP, NRLM and SCDH registers were respectively 45% (95%CI 40-51), 66% (95%CI 61-71) and 36.5% (95%CI 31-42). The estimated TB incidence rate was 15 cases per 100 000 (range 13-19/100 000), compared to the 4.9/100 000 rate officially notified. CONCLUSION: Adult TB incidence has been largely underestimated, and the TB burden is likely to be much higher than officially notified in our setting. A thorough review of the notification system should be carried out. The implementation of a network-based notification system and retraining of all relevant personnel is advised.

Fatal case of extensively drug-resistant Mycobacterium tuberculosis Beijing genotype infection in an injecting drug user, Athens, Greece, 2012.

We present the first fatal case of extensively drug-resistant tuberculosis (XDR-TB) in an injecting drug user (IDU) in Athens, Greece, co-infected with human immunodeficiency virus and hepatitis C virus and discuss the implications for public health. Despite immediate initiation of treatment, the patient's condition gradually deteriorated and he died 16 days after hospital admission because of multiple organ failure. The contact tracing investigation revealed no further infections among the patient's contacts.

Hepatitis B reactivation in a renal transplant patient due to a surface antigen mutant strain: a case report.

INTRODUCTION: Reactivation of hepatitis B virus (HBV) is a complication of immunosuppressive treatment in patients with a history of HBV exposure. CASE PRESENTATION: Herein we have reported a case of reactivation after renal transplantation in a 52-year-old male chronic HBV carrier who was treated with hepatitis B immunoglobulin (HBIg) prophylaxis immediately after transplantation in addition to cyclosporine, mycophenolate mofetil and prednisolone for maintenance immunosuppression. After application of rituximab, the patient developed clinical hepatitis with a high load of HBV DNA. Sequence analysis of the surface (S) antigen corresponding to the amino acid residues 101-186 (including the a-determinant region) revealed a genotype D mutant strain, subtype ayw3 with a single amino acid substitution D144E within the S gene. CONCLUSION: This case suggested that immunosuppressive treatment enhanced with rituximab promoted the emergence of an HBV mutant within the determinant region of the S antigen, which escaped HBIg immunoprophylaxis causing HBV reactivation in a kidney transplant recipient.

Diagnosis of tuberculosis and drug resistance: what can new tools bring us?

This is an exciting time for tuberculosis (TB) diagnostics. The technology for rapid diagnosis of TB and rifampicin (RMP) resistance in pulmonary sputum smear-positive specimens is well advanced, and assays have high specificity with good sensitivity. Nevertheless, the current sensitivity of TB detection means that these assays still cannot replace the standard diagnostic methods for TB or conventional drug susceptibility testing (DST). In extra-pulmonary specimens, the performance of molecular tools varies and should be considered separately for each specimen type. Evidence for the use of these assays for TB and drug resistance detection in individuals co-infected with TB and the human immunodeficiency virus (HIV) is limited. As the positive predictive value for RMP resistance reaches ≥ 90% only when the prevalence of RMP resistance in new TB patients is >15%, which is rare globally, many cases with such resistance will be false-resistant, emphasising the need for a secondary confirmative test. Similarly, increased (or incorrect) diagnosis of TB may compromise programme effectiveness by increasing the numbers of individuals requiring anti-tuberculosis treatment, unless it is carefully planned. For the future, 1) assays with greater sensitivity for TB detection are needed; 2) rapid diagnostics for paediatric TB are important, and there is a need for carefully designed studies, including those involving HIV-positive children; 3) more clinical data need to be obtained from longitudinal studies, especially related to the influence of rapid diagnostics on disease outcome; and 4) point-of-care tests using untreated sputum, blood or urine and little or no equipment would be of immeasurable benefit. Although great progress has been made, we are not there yet.

Trends in the epidemiology of childhood tuberculosis in Greece.

SETTING: A hospital referral center for childhood tuberculosis (TB). OBJECTIVE: To evaluate the epidemiological and clinical features of childhood TB in the Greater Athens area in the last decade. DESIGN: We retrospectively reviewed the medical records of patients aged <14 years treated for active TB between January 2000 and December 2009 at our pediatric TB clinic and compared the results with the patient turnover during the previous decade (1990-1999). Data concerning demographic and clinical characteristics were analyzed. RESULTS: A total of 321 children (median age 5.57 years, 157 males) with active TB were identified. About one third originated from areas where TB was previously recognized to be highly endemic. Twenty-three children (7%) had extra-pulmonary TB, and 61% of them originated from TB-endemic areas. Bacteriological confirmation was obtained in 40% of patients from whom specimens were obtained: 1 of 26 (3.8%) strains was multidrug-resistant. Most cases with drug-resistant Mycobacterium tuberculosis were noted among immigrant children. The average annual TB incidence was estimated at 5.37 per 100 000 for children aged <14 years in the Greater Athens area. Time trend analysis for the 20-year period revealed a significant reduction in the total number of TB cases (P = 0.002) and in TB among children from low-incidence countries (P < 0.0001). CONCLUSIONS: In our settings, active TB is decreasing among children of Greek origin; disease epidemiology and drug resistance is influenced by the increasing influx of immigrants from areas where the disease is highly prevalent.

Impact of the Gen-Probe Amplified MTD® Test on tuberculosis diagnosis in children.

OBJECTIVE: To evaluate the performance of the Gen-Probe Amplified MTD® Test (AMTD) for childhood tuberculosis (TB) diagnosis compared to conventional culture. DESIGN: We retrospectively studied 121 childhood cases (73 males; median age 7 years, range 1-16). Pulmonary samples (104/152, 68%) included gastric aspirates (n = 53), induced sputum samples (n = 43), bronchial aspirates and bronchoalveolar lavage (n = 8). Extra-pulmonary samples (48/152, 32%) included lymph nodes (n = 34) and other sterile fluids (n = 14). Specimens were examined using acid-fast bacilli (AFB) microscopy, AMTD and bacterial culture using BACTEC™ MGIT™ 960 and Löwenstein-Jensen (LJ) media. RESULTS: A clinical diagnosis of TB was made in 50/121 (41%) children (43/50 pulmonary disease). AFB microscopy was positive in 6%; Mycobacterium tuberculosis was recovered by culture from 16/50 (32%) and AMTD was positive in 29/50 (58%). AMTD sensitivity, specificity, positive predictive value and negative predictive value compared to culture were respectively 100%, 85%, 50% and 100%. For pulmonary vs. extra-pulmonary disease, the performance of AMTD compared to culture was respectively 100%, 77%, 46% and 100% vs. 100%, 97.5%, 75% and 100%. CONCLUSIONS: Nucleic acid amplification tests are more sensitive and very specific methods for the rapid detection of M. tuberculosis. The AMTD technique increases TB detection in children compared to conventional culture.

Tuberculosis in Greece: bacteriologically confirmed cases and anti-tuberculosis drug resistance, 1995-2009.

The Greek National Reference Laboratory for Mycobacteria is a major source of tuberculosis (TB)-related data for Greece, where the TB burden and epidemiology still need to be better defined. We present data regarding newly diagnosed TB cases and resistance to anti-TB drugs during the last 15 years in Greece. Although the total number of newly detected TB cases has declined, cases among immigrants are increasing. Resistance to first-line anti-TB drugs is widely prevalent, although stable or declining. The implementation of an efficient and effective countrywide TB surveillance system in Greece is urgently needed.

Viral agents of acute gastroenteritis in hospitalized children in Greece.

A 6-year study of stool samples from 4604 children hospitalized for acute gastroenteritis was conducted to investigate the role of enteric viruses as a cause of gastroenteritis in north-west Greece. Rotaviruses, noroviruses, adenoviruses and astroviruses were detected in 21.35%, 4%, 3.5% and 2.35%, respectively, by enzyme immunoassays and molecular techniques. Molecular techniques enhanced overall diagnostic efficacy by 2.5%, and by c. 10% each for rotavirus and adenovirus. Rotavirus was the leading cause of viral gastroenteritis, usually associated with severe illness. Mixed infections were found in 4.4% of positive specimens, and rotavirus plus astrovirus represented the most frequent co-infection (55.5%). This first study on the epidemiology of viral gastroenteritis in Greece shows that recent advances in the diagnosis of viral enteropathogens may have only marginal effects on overall diagnostic efficacy, and thus the impact of viral agents causing sporadic gastroenteritis in public health cannot be fully evaluated.

Measles and mumps immunity in Northern Greece, 2004-2007.

A cross-sectional study was conducted in order to determine the prevalence of mumps and measles antibodies in a representative sample of the general population in Northern Greece between January 2004 and May 2007. Overall, 900 healthy individuals participated in the study. The great majority were found to be protected against measles. The total protection rate against mumps was significantly less (87% versus 72%, respectively; p<0.01). Compared to all other age groups, statistically significantly lower protection rates were found in children younger than 1.5 years (p<0.01). The lowest rates of all adult groups were found in the age group of 21 to 30 years (86% and 68% for measles and mumps, accordingly). In conclusion, protection rates against both measles and mumps seem to be lower than expected in certain age groups, such as infants and young adults.

Cryptosporidium species causing acute diarrhoea in children in Antananarivo, Madagascar.

A 13-month study of children presenting with acute diarrhoeal disease at hospitals and rehydration clinics in Antananarivo, Madagascar, was undertaken between May 2004 and May 2005. Cryptosporidiosis accounted for diarrhoea in 12 (5.6%) of the 215 children investigated. Cases of cryptosporidiosis were detected only in the rainy season, and the median age of cases was 13.5 months (range=1 day-27 months). As 11 of the cases of cryptosporidiosis were caused by Cryptosporidium hominis and only one by C. parvum, most of the cases were probably the result of anthroponotic transmission. GP60/45/15 gene polymorphisms indicated that the causative pathogens were of subtypes Ia, Id, Ie and IIc.

Recombinant Sabin environmental isolates in Greece and Cyprus.

AIMS: Twenty-one polioviruses (PVs) Sabin strains were isolated from sewage treatment plants from Metamorphosis, Athens, Greece during the time period from May to October 1996, and from two other sites located at Nicosia and Limassol in Cyprus between April and December 2003 were retrospectively investigated for the detection of recombinant PVs. METHODS AND RESULTS: Three PVs isolates were found as tripartite recombinants, S3/S2/S1 in the 2C genomic viral region. The first recombination site S3/S2 was located close to the 5' end of 2C while the second recombination site S2/S1 was located towards the 3' end of 2C. Such recombination is a rare event producing a tripartite hybrid 2C protein. Three more PVs isolates were characterized as bipartite S2/S1 recombinants and one as S2/S3 bipartite recombinant. CONCLUSIONS: Detection of recombinant circulating vaccine-derived PVs (cVDPVs) is crucial, since increased transmissibility over that of the parental Sabin strains has been proposed to be the result of recombination events. SIGNIFICANCE AND IMPACT OF THE STUDY: Importation of recombinant cVDPVs evolved derivatives pose a serious threat to public health and environmental surveillance should be implemented during and after PVs eradication.

Membrane adsorption with direct cell culture combined with reverse transcription-PCR as a fast method for identifying enteroviruses from sewage.

We present a new approach for the detection and identification of enteroviruses concentrated and isolated from sewage. Samples were collected from two study sites located at Nicosia and Limassol sewage treatment plants in Cyprus. Viruses were adsorbed to cellulose nitrate membrane filters, cultured directly from the membrane filters by using the VIRADEN method, and identified by reverse transcription-PCR, followed by 5' untranslated region (5'-UTR) restriction fragment length polymorphism (RFLP) analysis and partial sequencing of the VP1 protein coding region. Initial subgrouping based on the HpaII restriction profile showed that all of the isolates except one belonged to the same genetic subcluster. Partial VP1 sequencing revealed that most isolates belonged to serotypes coxsackie B4 (42.5%) and coxsackie Alpha9 (30%), whereas coxsackie B2 (17.5%) and coxsackie B1 (3%) isolates were less frequently observed. One poliovirus type 2 isolate (2.5%) of vaccine origin was also found. The HpaII digests predicted the genetic subcluster for all isolates. They also accurately differentiated the isolates as nonpolio or polio isolates. This approach seems to be very promising for environmental surveillance of enterovirus circulation and epidemiology, with all of the significant effects that this entails for public health. Partial VP1 sequencing is efficient for molecular serotyping of enteroviruses, while 5'-UTR RFLP analysis with HpaII can also be considered an asset for the initial subclassification of enterovirus isolates.