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In Italy, rifampicin-resistant and MDR-TB were high in foreign-born persons, but decreased from 2009 to 2016

No information is currently available on the influence of injectable second-line drugs on treatment outcomes of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) patients. To investigate this issue, a large series of MDR- and XDR-TB cases diagnosed in Estonia, Germany, Italy and the Russian Federation (Archangels Oblast) between 1999 and 2006 were analysed. All study sites performed drug susceptibility testing for first- and second-line anti-TB drugs, laboratory quality assurance and treatment delivery according to World Health Organization recommendations. Out of 4,583 culture-confirmed cases, 240 MDR- and 48 XDR-TB cases had a definitive outcome recorded (treatment success, death, failure). Among MDR- and XDR-TB cases, capreomycin resistance yielded a higher proportion of failure and death than capreomycin-susceptible cases. Resistance to capreomycin was independently associated with unfavourable outcome (logistic regression analysis: odds ratio 3.51). In the treatment of patients with multidrug-resistant and extensively drug-resistant tuberculosis, resistance to the injectable drug capreomycin was an independent predictor for therapy failure in this cohort. As Mycobacterium tuberculosis drug resistance is increasing worldwide, there is an urgent need for novel interventions in the fight against tuberculosis.

To the Editor: Drug-resistant tuberculosis (TB) has been identified as a major problem in the former Soviet Union, and was recently surveyed in the Aral Sea regions of Dashoguz (Turkmenistan) and Karakalpakstan (Uzbekistan) (1). However, few data are available for the Caucasian region and published reports have focused mainly on prisons (2,3). We report a drug resistance survey for first- and second-line anti-TB drugs conducted in Abkhazia, a Caucasian region of 8,600 km2 with approximately 250,000 inhabitants, at the western end of Georgia on the Black Sea. The collapse of the Soviet Union lead to disruption of TB control activities in all Eastern bloc regions (4). In Abkhazia, the shortage and poor quality of drugs, self-medication, and poor adherence to the therapy became even more evident during the war with Georgia in 1993 and the international embargo that followed. A TB program based on the World Health Organization/International Union against Tuberculosis and Lung Disease (WHO/IUATLD) recommendations was initiated in Abkhazia with the support of Medecins Sans Frontieres (MSF) in 1999. In 2000, monitoring of drug resistance was started for new cases and previously treated case-patients. The study was performed in collaboration with the Guliripchi TB Hospital, MSF, and the Istituto Superiore di Sanita (ISS), a WHO/IUATLD Supranational Reference Laboratory for anti-TB drug resistance. Sputa were collected from all patients attending Guliripchi TB Hospital in Sukhumi, the capital of Abkhazia, from September 2000 to April 2004. Patients were either referred by their practitioners or came spontaneously because TB was suspected. Diagnosis, treatment, and hospitalization were provided free. Samples were treated as previously described (5). Of 489 sputa collected from individual patients, 447 were culture positive (246 from new case-patients and 201 from previously treated case-patients) and 42 were culture negative; of these, >90% showed a negative, doubtful, or 1+ smear result. Susceptibility to first-line (streptomycin, isoniazid, rifampin, and ethambutol) and second-line (kanamycin, ethionamide, capreomycin, cycloserine, p-aminosalicylic acid, and ofloxacin) drugs was determined by the proportion method on Middlebrook 7H10 agar. The critical concentrations used were streptomycin, 2 µg/mL; isoniazid, 0.2 µg/mL; rifampin, 1 µg/mL; ethambutol, 5 µg/mL; kanamycin, 5 µg/mL; ethionamide, 5 µg/mL; capreomycin, 10 µg/mL; p-aminosalicylic acid, 2 µg/mL; and ofloxacin, 2 µg/mL (6–8). Cycloserine was used at a concentration of 30 µg/mL (9). If a strain was resistant to >1 first-line drugs, the susceptibility to all second-line drugs was determined. Data on resistance to the first- and second-line drugs are given in the Table. The strains isolated from 35.8% of the new cases and 57.2% of the previously treated case-patients were resistant to >1 first-line drugs. The highest monoresistance was seen for isoniazid and streptomycin in both new and previously treated case-patients, while monoresistance to rifampin and ethambutol was low ( 1 second-line drugs. Table First-line and second line antituberculosis drug resistance in 447 Mycobacterium tuberculosis strains collected in Abkhazia from September 2000 to April 2004* Few data have been reported on drug resistance to first- and second-line drugs in the former Soviet Union and in the Caucasian region (1–4). Overall, in Abkhazia, monoresistance to isoniazid was higher than in Karakalpakstan and Dashoguz (1), while monoresistance to streptomycin was lower. MDR-TB in new and previously treated case-patients showed levels intermediate between these 2 regions. Resistance to kanamycin and ethionamide was 14.3% and 12.8%, respectively, while resistance to ofloxacin was low (1.5%). Fluoroquinolones have not been commonly used in Abkhazia and former regions of the Soviet Union. Currently, regimens for the treatment of MDR-TB in Abkhazia combine an intensive phase for a minimum of 6 months with at least 4 drugs to which the MTB strain is susceptible, including 1 parenteral agent and 1 fluoroquinolone (ofloxacin), followed by a continuation phase of at least 15 months with >3 drugs. This is the first survey reporting drug susceptibility data for MTB within the Caucasus. It indicates that the prevalence of MDR strains is similar to that in other central Asia regions (1). Our results are representative of the present situation in Abkhazia since sampling systematically covered all TB cases for the period examined. The Guliripchi TB Hospital of Sukhumi is the only TB treatment center in the region, and all cases were included in the study. Overall, our data show that second-line drug resistance is present in Abkhazia, particularly among cases with MDR, and suggest the adoption of strategies for access and correct use of second-line drugs (10).

In 1998 a network of 20 regional tuberculosis (TB) laboratories (the Italian Multicentre Study on Resistance to Antituberculosis drugs (SMIRA) network) was established in Italy to implement proficiency testing and to monitor the prevalence of drug resistance nationwide. The network managed 30% of all TB cases reported in Italy each year. The aim of the present report is to describe: 1) the accuracy of drug-susceptibility testing in the network; 2) the prevalence of drug resistance for the period 1998-2000. Data were collected from the network laboratories. Sensitivity to streptomycin and ethambutol increased from the first survey (1998-1999) to the second survey (2000) from 87.7 to 91.9%. Specificity, predictive values for resistance and susceptibility, efficiency and reproducibility were consistent in both surveys. In previously untreated cases, the prevalence of multidrug-resistance was the same in both surveys (1.2%), while a slight decrease from the first to the second survey was observed for monoresistance to rifampicin (from 0.8 to 0.4%) and isoniazid (from 2.9 to 2%). The significant association found between isoniazid resistance and immigration is a useful indicator for both clinicians managing individual tuberculosis cases and public health services planning control strategies.

Ethambutol (EMB) is in use worldwide as a first-line anti-tuberculosis drug and substitutions in codon 306 of the embB gene are the most common mutations found in EMB resistant Mycobacterium tuberculosis (MTB) strains. Pyrosequencing is a real time sequencing method able to rapidly detect mutations in a large number of samples. Using this technique we analyzed, in parallel with conventional sequencing, a 24 bp region of the embB gene of 28 MTB clinical isolates. Pyrosequencing efficiently identified all embB306 mutations, detecting three different single-base substitutions leading to 2 amino acid changes (Met to Val or Ile). Mutated embB alleles were detected in 2 multidrug-resistant (MDR) EMB-susceptible strains. Overall, our results demonstrated that the Pyrosequencing method efficiently recognizes mutations in embB in a very short time and represents a valid molecular method to detect mutations in the MTB embB306 region.

The recovery of Mycobacterium tuberculosis from sputa positive or negative for acid-fast bacilli that were stored for 17 ± 7 days and inoculated in the BACTEC MGIT 960 system (MGIT) was higher than that from sputa inoculated in Lowenstein-Jensen medium. MGIT is useful for isolation of M. tuberculosis from sputa subjected to long-term storage.

To the Editor: Twenty-three countries have reported >1 case of extensively drug-resistant tuberculosis (XDR TB) (1); however, information about XDR TB is still incomplete. In particular, the response of XDR TB to treatment in countries with low incidence is not known. We compared mortality rates from XDR TB with those from multidrug-resistant (MDR) TB. We analyzed data from all culture-confirmed TB cases diagnosed during 2003–2006 by the TB clinical reference centers in Italy (Sondalo, Milan, Rome) and Germany (Borstel, Grosshansdorf, Bad-Lippspringe) and reviewed original clinical records. Drug susceptibility testing for first- and second-line anti-TB drugs was performed according to World Health Organization (WHO) recommendations by quality-assured laboratories and retested at WHO Supranational Reference Laboratories (Rome/Milan; Borstel) (2–4). XDR TB was defined as resistance to at least rifampin and isoniazid (MDR TB definition) in addition to any fluoroquinolone and >1 of 3 injectable anti-TB drugs (capreomycin, kanamycin, amikacin) (3). Characteristics of MDR TB and XDR TB cases were compared by χ2 test (categorical variables), Student t test (admission days), and Kaplan-Maier curve (sputum smear, culture conversion), where appropriate. Of 2,888 culture-positive TB cases analyzed (Italy 2,140, Germany 748), 126 (4.4%) were MDR (Italy 83, Germany 43) and 11 (0.4%) were XDR (Italy 8, Germany 3). We estimate that the TB cases analyzed represent 24% of culture-positive cases reported in Italy (69.7% of MDR) and 4.2% of those reported in Germany (12.6% of MDR). XDR TB was diagnosed in each year of the study. All 11 XDR TB patients were receiving retreatment, and of the 126 MDR TB patients, 74 (58.7%) were receiving retreatment. All XDR TB patients were HIV seronegative; and of 109 MDR TB patients tested for HIV, 10 (9.2%) were HIV seropositive. Details about previous treatment regimens, drug resistance, and duration of treatment of XDR TB patients are summarized in the Appendix Table. XDR TB patients were significantly more likely than MDR TB patients to be resistant to all first-line drugs (8/11 vs. 36/126, p

Currently, no information is available on the effect of resistance/susceptibility to first-line drugs different from isoniazid and rifampicin in determining the outcome of extensively drug-resistant tuberculosis (XDR-TB) patients, and whether being XDR-TB is a more accurate indicator of poor clinical outcome than being resistant to all first-line anti-tuberculosis (TB) drugs. To investigate this issue, a large series of multidrug-resistant TB (MDR-TB) and XDR-TB cases diagnosed in Estonia, Germany, Italy and the Russian Federation during the period 1999-2006 were analysed. Drug-susceptibility testing for first- and second-line anti-TB drugs, quality assurance and treatment delivery was performed according to World Health Organization recommendations in all study sites. Out of 4,583 culture-positive TB cases analysed, 361 (7.9%) were MDR and 64 (1.4%) were XDR. XDR-TB cases had a relative risk (RR) of 1.58 to have an unfavourable outcome compared with MDR-TB cases resistant to all first-line drugs (isoniazid, rifampicin ethambutol, streptomycin and, when tested, pyrazinamide), and an RR of 2.61 compared with "other" MDR-TB cases (those susceptible to at least one first-line anti-TB drug among ethambutol, pyrazinamide and streptomycin, regardless of resistance to the second-line drugs not defining XDR-TB). The emergence of extensively drug-resistant tuberculosis confirms that problems in tuberculosis management are still present in Europe. While waiting for new tools which will facilitate management of extensively drug-resistant tuberculosis, accessibility to quality diagnostic and treatment services should be urgently ensured and adequate public health policies should be rapidly implemented to prevent further development of drug resistance.

Recovery of Mycobacterium tuberculosis from sputa treated with cetyl-pyridinium chloride (CPC) and stored for 20 ± 9 days was significantly higher than that from sputa that were untreated and processed by the N -acetyl- l -cisteine-NaOH method. Addition of CPC is useful for isolation of M. tuberculosis from sputa subjected to long-term storage received from remote areas of the world.

Although multidrug-resistant (MDR) tuberculosis (TB) is a major public health problem in Eastern Europe, the factors contributing to emergence, spread and containment of MDR-TB are not well defined. Here, we analysed the characteristics of drug-resistant TB in a cross-sectional study in Abkhazia (Georgia) between 2003 and 2005, where standard short-course chemotherapy is supplemented with individualized drug-resistance therapy. Drug susceptibility testing (DST) and molecular typing were carried out for Mycobacterium tuberculosis complex strains from consecutive smear-positive TB patients. Out of 366 patients, 60.4% were resistant to any first-line drugs and 21% had MDR-TB. Overall, 25% of all strains belong to the Beijing genotype, which was found to be strongly associated with the risk of MDR-TB (OR 25.9, 95% CI 10.2-66.0) and transmission (OR 2.8, 95% CI 1.6-5.0). One dominant MDR Beijing clone represents 23% of all MDR-TB cases. The level of MDR-TB did not decline during the study period, coinciding with increasing levels of MDR Beijing strains among previously treated cases. Standard chemotherapy plus individualized drug-resistance therapy, guided by conventional DST, might be not sufficient to control MDR-TB in Eastern Europe in light of the spread of "highly transmissible" MDR Beijing strains circulating in the community.

Mutations of rpoB associated with rifampin resistance were studied in 37 multidrug-resistant (MDR) clinical strains of Mycobacterium tuberculosis isolated in Italy. At least one mutated codon was found in each MDR strain. It was always a single-base substitution leading to an amino acid change. Nine different rpoB alleles, three of which had not been reported before, were found. The relative frequencies of specific mutations in this sample were different from those previously reported from different geographical areas, since 22 strains (59.5%) carried the mutated codon TTG in position 531 (Ser→Leu) and 11 (29.7%) had GAC in position 526 (His→Asp).

The antimicrobial peptide protegrin-1 (PG-1) inhibited the growth in vitro of drug-susceptible and multidrug-resistant Mycobacterium tuberculosis; a lower activity was shown by human beta-defensin-1 (HBD-1) against both strains. The combination of PG-1 or HBD-1 with isoniazid significantly reduced M. tuberculosis growth in comparison with the peptides or isoniazid alone.

Controversial results have been obtained in studies of the effect of Mycobacterium tuberculosis on human immunodeficiency virus type 1 (HIV-1) replication in cells of the macrophage lineage. In the present study, monocyte-derived macrophages (MDMs), previously incubated for 2 days with heat-inactivated M. tuberculosis, were infected with HIV-1. M. tuberculosis consistently inhibited viral replication, and a similar result also was observed in the presence of supernatants from M. tuberculosis-stimulated MDMs, which indicates that this effect was mediated by soluble factors. Although CCR5-binding chemokines were induced by M. tuberculosis stimulation, the results of neutralization experiments indicated that it is unlikely that they were responsible for viral suppression. Inhibition occurred mainly after viral entry (demonstrated by use of a vesicular stomatitis virus G-pseudotyped HIV-1 and by analysis of HIV-1 early and late reverse-transcription products). Therefore, M. tuberculosis-induced factors may inhibit in vitro HIV-1 replication in macrophages by affecting an early postentry step in the HIV-1 cycle.

The bacterial growth and the production of tumor necrosis factor alpha (TNF-α) and TNF receptors (TNF-Rs) in the spleen and blood of BALB/c mice challenged with Mycobacterium avium complex (MAC) were monitored. Infection developed in two phases: the first, up to day 21, was associated with rapid MAC multiplication in the spleen and a drop in the mycobacteremia, and the second was associated with control of the infection in both compartments. In the spleen, TNF-α and TNF-RII mRNA levels peaked on day 21 and then slowly decreased; however, no increase in the level of TNF-RI mRNA was observed throughout these experiments. The level of circulating soluble TNF-RII (sTNF-RII) was transiently increased after day 21. In a model in which overproduction of bioactive TNF-α was triggered in response to a second infection with MAC, an increased production of sTNF-RII by cultured splenocytes was also observed. Administration of an antagonist anti-TNF-RII monoclonal antibody (MAb 6G1) to infected mice inhibited the bacterial growth in the spleen, suggesting that the TNF-RII and/or sTNF-RII was functionally involved in the mechanisms that control the infection. Overall, these observations suggest that upregulation of TNF-RII or sTNF-RII contributes to modulation of the TNF-α antibacterial activity in MAC infections.