Paola Molicotti

Detection and Isolation of Mycobacterium avium Subspecies paratuberculosis from Intestinal Mucosal Biopsies of Patients with and without Crohn's Disease in Sardinia

OBJECTIVES:Sardinia is an island community of 1.6 million people. There are also about 3.5 million sheep and one hundred thousand cattle in which Johnes disease and Mycobacterium avium subspecies paratuberculosis infection are endemic. The present study was designed to determine what proportion of people in Sardinia attending for ileocolonoscopy with or without Crohns disease were infected with this pathogen.METHODS:Mycobacterium avium subspecies paratuberculosis was detected by IS900 PCR on DNA extracts of fresh intestinal mucosal biopsies as well as by isolation in culture using supplemented MGIT media followed by PCR with amplicon sequencing.RESULTS:Twenty five patients (83.3%) with Crohns disease and 3 control patients (10.3%) were IS900 PCR positive (p = 0.000001; Odds ratio 43.3). Mycobacterium avium subspecies paratuberculosis grew in cultures from 19 Crohns patients (63.3%) and from 3 control patients (10.3%) (p = 0.00001; Odds ratio 14.9). All patients positive by culture had previously been positive by PCR. Mycobacterium avium subspecies paratuberculosis first appeared in the liquid cultures in a Ziehl Neelsen (ZN) staining negative form and partially reverted through a rhodamine-auramine positive staining form to the classical ZN positive form. This resulted in a stable mixed culture of all 3 forms illustrating the phenotypic versatility of these complex chronic enteric pathogens.CONCLUSIONS:Mycobacterium avium subspecies paratuberculosis was detected in the majority of Sardinian Crohns disease patients. The finding of the organism colonizing a proportion of people without Crohns disease is consistent with what occurs in other conditions caused by a primary bacterial pathogen in susceptible hosts.

Antibacterial activity of ozonized sunflower oil (OLEOZON)

L.A. SECHI, I. LEZCANO, N. NUNEZ, M. ESPIM, I. DUPRÈ, A. PINNA, P. MOLICOTTI, G. FADDA AND S. ZANETTI. 2001.

Evaluation of the Antimicrobial Properties of the Essential Oil of Myrtus communis L. against Clinical Strains of Mycobacterium spp.

Mycobacterium tuberculosis is the etiological agent of tuberculosis. The World Health Organization has estimated that 8 million of people develop active TB every year and the situation is complicated by an increase of Mycobacterium tuberculosis strains resistant to drugs used in antitubercular therapy: MDR and XDR-TB. Myrtle leaf extracts, used as an antiseptic in Sardinian traditional medicine, have strong antibacterial activity as several investigations showed. In this study we investigated the antimicrobial properties of the essential oil of Myrtus communis against clinical strains of M. tuberculosis and M. paratuberculosis.

Methylated HBHA Produced in M. smegmatis Discriminates between Active and Non-Active Tuberculosis Disease among RD1-Responders

Background A challenge in tuberculosis (TB) research is to develop a new immunological test that can help distinguish, among subjects responsive to QuantiFERON TB Gold In tube (QFT-IT), those who are able to control Mtb replication (remote LTBI, recent infection and past TB) from those who cannot (active TB disease). IFN-γ response to the Heparin-binding-hemagglutinin (HBHA) of Mtb has been associated with LTBI, but the cumbersome procedures of purifying the methylated and immunological active form of the protein from Mtb or M. bovis Bacillus Calmette et Guerin (BCG) have prevented its implementation in a diagnostic test. Therefore, the aim of the present study was to evaluate the IFN-γ response to methylated HBHA of Mtb produced in M. smegmatis (rHBHAms) in individuals at different stages of TB who scored positive to QFT-IT. Methodology/Principal Findings 87 individuals at different stages of TB who scored positive to QFT-IT were selected. IFN-γ response to in vitro whole blood stimulation with rHBHAms was evaluated by short-term and long-term tests and detected by ELISA or flow cytometry. We demonstrated that the IFN-γ response to rHBHAms is mediated by CD4+ T-cells with an effector-memory phenotype. This response, evaluated by short-term-tests, is significantly lower in active TB than in remote LTBI (p = 0.0010) and past TB (p = 0.0152). These results were confirmed by long-term tests. The qualitative data confirmed that IFN-γ responses higher than the cut-off point identified by ROC analysis are associated with the status of non-active disease. Conclusions In this study we show that the T-cell response to a recombinant and methylated HBHA of Mtb produced in M. smegmatis is useful to discriminate between active and non-active TB disease among those responsive to QFT-IT in a whole blood system. Further studies are needed to improve the accuracy of the assay.

Rapid identification of cutaneous infections by nontubercular mycobacteria by polymerase chain reaction-restriction analysis length polymorphism of the hsp65 gene

Background  Nontubercular mycobacteria (NTM) may cause cutaneous infections which are difficult to interpret due to the variability of the clinical manifestations. This study involved eight patients (four men and four women) with primary cutaneous infections caused by NTM; the skin lesions included dermo‐hypodermal abscesses, suppurative granulomas, and papulonodules localized on the legs, arms, hands, and face. The general condition of the patients was relatively good and they were not immunosuppressed.

Performance of QuantiFERON-TB Testing in a Tuberculosis Outbreak at a Primary School

This study compared the effectiveness of the QuantiFERON-TB Gold (QFT) assay with the Mantoux tuberculin skin test to detect Mycobacterium tuberculosis infection in 29 children during a school outbreak of tuberculosis. Of the 21 children with M. tuberculosis infection, 11 had a radiograph suggestive of the infection. The QFT assay was positive in all 21 of the children, and the Mantoux test was negative at first testing in 2 children (1 of whom was the sentinel case). The findings demonstrate that the QFT test is extremely useful in accurately identifying infected and uninfected children, permitting rapid intervention.

Identification of mycobacterial infections in wild boars in Northern Sardinia, Italy

During a six-month period a region of Northern Sardinia was monitored to check the presence of mycobacterial infections in wild boars. Forty-eight serum and 229 biopsy samples were collected from different animals and examined by both traditional diagnostic techniques (culture, bacterioscopic and molecular tests) and enzyme-linked immunosorbent assay (ELISA). The latter was used to determine the antibody response against both methylated and nonmethylated Heparin-Binding Haemagglutinin (HBHA) protein. Nine mycobacterial strains were isolated: three M. avium ssp. paratuberculosis (Map), three M. avium, one M. interjectum and two M. scrofulaceum strains. By PCR, only one animal was positive for M. bovis, whereas 10 animals were positive for Map. Out of the 48 sera tested, 19 showed a good humoral response to methylated HBHA and 17 to nonmethylated HBHA. Our data provide new information on the prevalence of mycobacterial infection among wild boars in Northern Sardinia and suggest that a more effective program should be developed to monitor mycobacterial infections in the wild animal population.

Molecular Basis of Rifampin and Isoniazid Resistance in Mycobacterium bovis Strains Isolated in Sardinia, Italy

ABSTRACT Fourteen of 22 (68%) Mycobacterium bovis strains isolated from cattle in Sardinia were found to be resistant to rifampin and isoniazid. Analysis of the rpoB and the katG, oxyR-ahpC, and inhA gene regions of these strains was performed in order to investigate the molecular basis of rifampin and isoniazid resistance, respectively. The most frequent mutation, encountered in 6 of 10 strains (60%), was in the rpoBgene; it occurred, at codon position 521 and resulted in leucine changed to proline. This suggests that codon 521 may be important for the development of rifampin resistance in M. bovis. Resistance to isoniazid is associated in Mycobacterium tuberculosis with a variety of mutations affecting one or more genes. Our results confirm the difficulty of interpreting the sequence variations observed in clinical strains of M. bovis. M. bovis strains isolated from the same geographic area showed similar mutations within the genes responsible for rifampin and isoniazid resistance. Our results represent the first study to elucidate the molecular genetic basis of drug resistance in M. bovis isolated from cattle.

Cost-effectiveness in the diagnosis of tuberculosis: choices in developing countries

Tuberculosis remains one of the major causes of global death from a single infectious agent. This situation is worsened by the HIV/AIDS pandemic because one-third of HIV/AIDS patients are co-infected with Mycobacterium tuberculosis. Failure to control the spread of tuberculosis is largely due to our inability to detect and treat all infectious cases of pulmonary tuberculosis in a timely manner, allowing continued M. tuberculosis transmission within communities. Diagnosis of tuberculosis can be made using indirect and direct methods. The indirect tests, such as interferon-gamma release assays, provide a new diagnostic method for M. tuberculosis infection, but do not discriminate between infection and active disease. The most common direct method for diagnosing TB worldwide is sputum smear microscopy (developed more than 100 years ago), where bacteria are observed in sputum samples examined under a microscope. In countries with more developed laboratory capacities, cases of tuberculosis may also be diagnosed using culture methods (the current gold standard) or, increasingly, using rapid molecular tests. In this review, we discuss the traditional methods for the diagnosis of tuberculosis. We also discuss other inexpensive assays that can be used to detect the presence of M. tuberculosis.

Tuberculosis Screening before Anti–Hepatitis C Virus Therapy in Prisons

To the Editor: Prisons represent a crucial setting for tuberculosis (TB) control. Worldwide, reported TB rates for correctional system populations have been 10–100× higher than rates for the local civilian populations, and TB outbreaks with a high number of TB multidrug-resistant cases have been documented (1,2). Prisons are known as social and sanitary pathology reservoirs in which TB is often associated with chronic infectious diseases caused by HIV, hepatitis B virus (HBV), or hepatitis C virus (HCV) (2). HCV prevalence among inmates is 30%–40% (range 2%–58%), which is higher than that in the general population and is related to injection drug use (3). For these reasons, effective anti-HCV therapeutic approaches are recommended by national and international guidelines for decreasing illness, death rates, and reservoirs of infection in prisons (4,5). The standard of care for patients with chronic hepatitis C infection is represented by pegylated interferon-α (Peg-IFN) and ribavirin. These drugs determine complex antiviral, immunomodulatory, and antiproliferative actions, which can cause serious side effects such as leukopenia/neutropenia and alterations in the cytokine network (3). Although severe cellular immunodeficiency can often facilitate the development of many infections, only 4 clinical cases of TB in patients undergoing HCV antiviral therapy have been described in the literature (6–8), and only 1 of these was clearly described as a TB reactivation (7). We describe a case of pulmonary TB reactivation during therapy with Peg-IFN and ribavirin in a 44-year-old white male inmate, affected by genotype 1b/4a chronic hepatitis C. After prison admission in 2009, he underwent routine screening tests for infectious diseases, which indicated HCV antibody, HBV surface antibody, HBV core IgG antibody, and tuberculin skin test positivity. Results of chest radiograph and HIV screening were negative. His previous history involved injection drug use, smoking, and alcohol consumption. Anti-HCV therapy of directly observed administration of Peg-INF α-2a (180 µg/wk) and ribavirin (1,200 mg/d) was started. During therapy, the patient had only mild musculoskeletal pain and temporary irritability. During the 12th week of treatment, HCV-RNA decreased by 1 log10; therefore, the ribavirin dose was increased to 1,600 mg per day. Even after the therapy modification, no virologic suppression was found. Although during the 33rd week of therapy the patient had weakness, cough, and 2 episodes of hemoptysis, the results of a physical examination were unremarkable. Therapy was immediately discontinued. Sputum specimens collected on 3 consecutive days were positive for acid-fast bacilli. Nucleic acid amplification assays and cultures performed on mycobacteria growth indicator tube (Bactec MGIT; Becton Dickinson, Franklin Lakes, NJ, USA) and on Lowenstein-Jensen medium were positive for Mycobacterium tuberculosis isolates that later showed sensitivity to streptomycin, isoniazid, rifampin, and ethambutol. The patient was isolated at the Institute of Respiratory Diseases, University of Sassari–Faculty of Medicine, Sassari, Italy. A chest radiograph showed opacity in the upper right lung, and a high-resolution computed tomography scan (Figure) showed multiple lesions that were considered compatible with TB. CD4+ cell count (52.4%; 669 cells/mm3) was within reference range. Figure Computed tomography image of chest of patient with tuberculosis after anti–hepatitis C virus therapy. A parenchymal distortion 32 mm in diameter is shown in the upper right lung with initial central excavation 10 mm in diameter. Similar lesions ... TB treatment with rifampin, isoniazid, pyrazinamide, and ethambutol with pyridoxine was started. After 4 weeks of therapy, 3 sputum specimens were negative for acid-fast bacilli, but a bacterial culture was still positive; mycobacteria indicator growth tube culture was negative after 5 weeks. The interaction process between the IFN-α/β system and M. tuberculosis is not well known; nevertheless, Peg- IFN, alone and in combination with ribavirin, is considered potentially immunosuppressive (4,9). Immunodeficiency caused by Peg-IFNs and ribavirin may cause lower leukopenia/lymphopenia values than expected during anti-HCV treatment and may also lower CD4+ cell count and function (10). In the patient reported here, CD4+ cell count was within the reference range, and lung TB with excavations developed after 34 weeks of therapy. Before TB diagnosis, the patient had not shown any signs or symptoms of other infections and had not mentioned serious adverse effects from Peg-IFN and ribavirin treatment. However, the initial symptoms of TB and the common side effects of Peg-IFN therapy can be similar, which could have led to a delay in the diagnosis of TB. In conclusion, even if only a few cases of active TB have been reported in the literature, it is well known that standard anti-HCV treatment increases the risk for infections. A high proportion of patients with positive purified protein derivative results, isolation of >30% of multidrug-resistant strains of M. tuberculosis, and high prevalence of HCV antibody are concomitant among inmates. These data, together with current recommendations for increasing use of Peg-IFN and ribavirin in marginalized populations in correctional facilities, show the need to consider TB risk before starting HCV antiviral therapy. The management of simultaneous HCV and M. tuberculosis infections in prisons presents particular difficulties and pitfalls to overcome. In prisons, the clinical history of inmates should be carefully evaluated, a tuberculin skin test or Quantiferon TB in Tube test (Cellestis, Melbourne, Australia) should be performed, and, if those results are positive, a chest radiograph should be taken. Before receiving Peg-IFN, purified protein derivative–positive patients should receive anti-TB chemoprophylaxis. The case described here underscores the need for a careful and multidisciplinary evaluation of inmate patients for latent TB before administration of Peg-IFN and ribavirin therapy, thus avoiding reactivation.