“Mycobacterium tilburgii,” a Newly Described,Uncultivated Opportunistic Pathogen

Molecular methods are increasingly used to identify pathogens that are difficult to cultivate. We report a case of disseminated infection with “Mycobacterium tilburgii,” a proposed species that has never been cultivated. The case illustrates the diagnostic utility of sequence analysis of the 16S rRNA gene directly from clinical specimens.Molecular methods are increasingly used to detect pathogens that are difficult to cultivate or have thus far not been cultivated. Amplification and sequencing of the 16S rRNA gene directly from clinical specimens can produce data that enable rapid detection of such organisms and their identification to the species level (2, 5). “Mycobacterium tilburgii” is a proposed species that has not been cultivable and has been reported in only four previous patients. It was first described by Buiting et al. in a previously healthy woman with acid-fast smear-positive infection of her bowels and bladder (1, 9). The organism had a unique 16S rRNA gene sequence and was named for the Dutch city in which it was first detected, Tilburg. The only other published cases are those of patients with human immunodeficiency virus (HIV)/AIDS who developed enteritis (8, 9) and pulmonary nodules (6) due to noncultured mycobacterial species. Comparison of the 16S rRNA gene sequences against those in GenBank identified the organisms in all three cases as “M. tilburgii”.We report an additional case of disseminated “M. tilburgii” infection in an HIV-negative patient seen at the National Institutes of Health (NIH).A 53-year-old man from New Hampshire with a history of sarcoidosis and atherosclerotic cardiovascular disease was admitted to the NIH for evaluation of abdominal pain and diarrhea.The patient had undergone a partial right lower lobectomy 13 years earlier for pulmonary nodules diagnosed histologically as sarcoidosis. He was treated with prednisone for 1 year with resolution of the residual nodules. He also had recurrent esophageal strictures treated with endoscopic dilation.The patient was in stable health until a year prior to admission, when he developed daily episodes of severe nonradiating periumbilical abdominal pain and intermittent watery diarrhea with occasional streaks of blood. Esophagogastroduodenoscopy and colonoscopy findings were normal, but small bowel enteroscopy demonstrated yellow duodenal and jejunal plaques. Acid-fast smear of the biopsy samples of the plaques revealed numerous acid-fast bacilli, but cultures were negative.He was treated empirically for disseminated Mycobacterium avium complex infection with clarithromycin, ethambutol, and rifabutin with transient improvement of his symptoms. A computed tomography (CT) scan performed for recurrent abdominal pain 3 months before admission to the NIH demonstrated a mesenteric abscess. A needle biopsy yielded pus that had a positive acid-fast smear but was again culture negative. Following the biopsy, he was treated with clarithromycin, rifabutin, and amikacin. His CD4⁺ T-cell counts ranged from 140 to 350/μl (normal, >400/μl). He was repeatedly negative for HIV type 1 (HIV-1) and HIV-2 by enzyme-linked immunosorbent assay. He had lost more than 14 kg over a year. The patient had fatigue but no fevers, chills, or night sweats. He had never traveled outside North America and had a past history of heavy tobacco and alcohol use.On examination at NIH, he was afebrile with normal vital signs. He appeared well nourished but mildly ill. He was barrel chested and had diffusely decreased breath sounds with scattered expiratory wheezes. There was moderate tenderness to palpation at the left periumbilical region with no palpable masses or liver edge. The spleen was palpable just below the left costal margin. Hemoccult-positive brown stool was in the rectal vault. He had mild peripheral clubbing and leukonychia. Cardiac, neurologic, and lymph node examination results were normal.His erythrocyte sedimentation rate was 22 mm/h. His white blood cell count was 5,600/μl. He was negative for HIV-1 and -2 by enzyme-linked immunosorbent assay. A CT scan showed several large, low-density, ring-enhancing masses in the mesentery, the largest of which was 5.1 by 3.6 cm and involved the small bowel wall (Fig. ​(Fig.1).1). Upper endoscopy with push enteroscopy found scattered yellow plaques in the jejunum and duodenum (Fig. ​(Fig.2).2). After failed percutaneous drainage, the patient underwent a laparotomy. He had a thickened, nodular jejunal mucosa with matted purulent mesenteric lymph nodes, the largest of which measured 4.5 cm. A partial small bowel resection, including removal of the associated mesentery, was performed.Open in a separate windowFIG. 1.Abdominal CT scan of our patient, demonstrating the largest of several mesenteric masses (arrow), measuring 5.1 by 3.6 cm.Open in a separate windowFIG. 2.Endoscopic photograph of the jejunal wall, which had scattered yellow plaques.Biopsy samples from the small bowel abscesses showed granulomatous changes and numerous acid-fast organisms. Cultures remained sterile. The patient was treated with clarithromycin, ethionamide, moxifloxacin, and subcutaneous gamma interferon (50 μg/m² three times weekly). His symptoms and CT findings resolved over several months. CT scans 1, 2, and 3 years postoperatively showed continued resolution of his abscesses. After 2 years, gamma interferon was discontinued and the patient has remained well on secondary prophylaxis with moxifloxacin and clarithromycin.A total of 14 clinical specimens were submitted for mycobacterial culture (two each of blood, sputum, and a mesenteric abscess; one each of urine, stool, and a left foot ulcer; and biopsy samples of the colon, small bowel, duodenum, skin, and a mesenteric lymph node). Variable numbers of acid-fast bacilli were seen, by auramine-rhodamine staining (Becton Dickinson, Sparks, MD), on the direct smears of the colonic, jejunal, and duodenal biopsy samples and of the lymph node and mesenteric abscess materials.All samples were plated on BACTEC 12B, Middlebrook 7H11 agar, and Mitchison 7H11 agar (Middlebrook 7H11 with carbenicillin, polymyxin B, amphotericin B, and trimethoprim; Remel, Lenexa, KS). In an attempt to isolate fastidious mycobacterial species known to have specific nutritional requirements, material from some specimens was inoculated onto one or more of the following media: Lowenstein-Jensen slants, Herrold''s egg yolk agar with mycobactin J, sheep blood agar, horse blood agar, and charcoal yeast extract agar (Remel). Multiple BACTEC 12B vials were also inoculated for each specimen; various supplements were added to individual vials, including reconstituting fluid (BD), egg yolk (BD), mycobactin J (Allied Monitor, Fayette, MO), and hemin solution (Sigma, St. Louis, MO). Solid media were incubated either at 30°C in ambient air or at 35°C in 5 to 8% CO₂; BACTEC 12B vials were incubated at both 30 and 35°C. Cultures were incubated for 10 to 16 weeks and remained negative, except for single colonies of Mycobacterium mucogenicum, Mycobacterium gordonae, and Acremonium species from the colonic biopsy sample. These organisms were not considered pathogens in this patient, given the source, the species, and the fact that only a single colony of each was isolated.For DNA extraction, amplification, and sequence analysis, organismal DNA was extracted from mesenteric abscess fluid by a modification of a previously described procedure (4). Briefly, the fluid sample was lysed in guanidinium thiocyanate buffer. The DNA was extracted with phenol-chloroform-isoamyl alcohol, purified with the Gene Clean II kit (MP Biomedicals, Solon, OH), and eluted with Tris-EDTA buffer.A 1,236-bp fragment of the 16S rRNA gene was amplified and sequenced as previously described (3, 4). Sequences were assembled by SeqMan II software (DNAstar, Madison, WI). Related sequences were identified by the Basic Local Alignment Search Tool (BLAST; National Center for Biotechnology Information, Bethesda, MD). No material other than the abscess fluid was available for molecular analysis.Numerous specimens from this patient were positive for acid-fast bacilli, but no pathogens could be isolated despite the use of a variety of culture methodologies. The sequence of the 1,236-bp region of the 16S rRNA gene amplified from the abscess material contained no ambiguous bases and showed 100% similarity to the sequence of “M. tilburgii,” a proposed species submitted to GenBank in 1995 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"Z50172","term_id":"1480398","term_text":"Z50172"}}Z50172). Given the perfect sequence match and the failure of any likely pathogen to grow from this patient''s specimens, it is most probable that “M. tilburgii” was the cause of his infection.“M. tilburgii” has been reported four times previously as a human pathogen. The original report described a patient with cutaneous anergy but no other evidence of immune compromise (1). The three subsequent cases occurred in profoundly immunosuppressed patients with HIV/AIDS. Our patient is similar to the one in the original report; he had no known immune defect, the infection was visceral, and therapy was successful. It is possible that his earlier diagnosis of sarcoidosis was due to an abnormal immune reaction to this organism. However, his improvement with steroids makes this unlikely. Steroid therapy could have put him at risk for acquiring this infection, but that seems improbable, as it preceded the infection by many years.The molecular study of organisms difficult or impossible to cultivate, or difficult to identify by phenotypic testing, has led to the recognition of many new microbial species, including new species of mycobacteria (7). “M. tilburgii” is representative of organisms currently identifiable only through molecular techniques.There are at least two cogent reasons for thinking that “M. tilburgii” was, in fact, a pathogen in this case. First, numerous acid-fast organisms were seen in the biopsy samples and abscess material, so one would have predicted the pathogen to be a species of Mycobacterium. Second, the “M. tilburgii” sequence has only been reported four times previously and only from human patient material, not from animal or environmental sources.In addition to providing a fifth report of “M. tilburgii” infection, this case illustrates the diagnostic utility of organismal DNA extraction from clinical lesions, particularly those in which organisms can be visualized but from which no likely pathogen can be cultivated, followed by amplification and sequencing of an appropriate genetic target.     

Coordination of gaze and hand movements for tracking and tracing in 3D

In this study we have investigated movements in three-dimensional space. Since most studies have investigated planar movements (like ellipses, cloverleaf shapes and “figure eights”) we have compared two generalizations of the two-thirds power law to three dimensions. In particular we have tested whether the two-thirds power law could be best described by tangential velocity and curvature in a plane (compatible with the idea of planar segmentation) or whether tangential velocity and curvature should be calculated in three dimensions. We defined total curvature in three dimensions as the square root of the sum of curvature squared and torsion squared. The results demonstrate that most of the variance is explained by tangential velocity and total curvature. This indicates that all three orthogonal components of movements in 3D are equally important and that movements are truly 3D and do not reflect a concatenation of 2D planar movement segments.In addition, we have studied the coordination of eye and hand movements in 3D by measuring binocular eye movements while subjects move the finger along a curved path. The results show that the directional component and finger position almost superimpose when subjects track a target moving in 3D. However, the vergence component of gaze leads finger position by about 250 msec. For drawing (tracing) the path of a visible 3D shape, the directional component of gaze leads finger position by about 225 msec, and the vergence component leads finger position by about 400 msec. These results are compatible with the idea that gaze leads hand position during drawing movement to assist prediction and planning of hand position in 3D space.     

Hippocampal NPY gene transfer attenuates seizures without affecting epilepsy-induced impairment of LTP

Recently, hippocampal neuropeptide Y (NPY) gene therapy has been shown to effectively suppress both acute and chronic seizures in animal model of epilepsy, thus representing a promising novel antiepileptic treatment strategy, particularly for patients with intractable mesial temporal lobe epilepsy (TLE). However, our previous studies show that recombinant adeno-associated viral (rAAV)-NPY treatment in naive rats attenuates long-term potentiation (LTP) and transiently impairs hippocampal learning process, indicating that negative effect on memory function could be a potential side effect of NPY gene therapy.Here we report how rAAV vector-mediated overexpression of NPY in the hippocampus affects rapid kindling, and subsequently explore how synaptic plasticity and transmission is affected by kindling and NPY overexpression by field recordings in CA1 stratum radiatum of brain slices. In animals injected with rAAV-NPY, we show that rapid kindling-induced hippocampal seizures in vivo are effectively suppressed as compared to rAAV-empty injected (control) rats. Six to nine weeks later, basal synaptic transmission and short-term synaptic plasticity are unchanged after rapid kindling, while LTP is significantly attenuated in vitro. Importantly, transgene NPY overexpression has no effect on short-term synaptic plasticity, and does not further compromise LTP in kindled animals. These data suggest that epileptic seizure-induced impairment of memory function in the hippocampus may not be further affected by rAAV-NPY treatment, and may be considered less critical for clinical application in epilepsy patients already experiencing memory disturbances.     

Neuroimaging as a tool for pain diagnosis and analgesic development

Neuroimaging makes it possible to study pain processing beyond the peripheral nervous system, at the supraspinal level, in a safe, noninvasive way, without interfering with neurophysiological processes. In recent years, studies using brain imaging methods have contributed to our understanding of the mechanisms responsible for the development and maintenance of chronic pain. Moreover, neuroimaging shows promising results for analgesic drug development and in characterizing different types of pain, bringing us closer to development of mechanism-based diagnoses and treatments for the chronic pain patient.     

Diagnostic and prognostic validity of the human papillomavirus E6/E7 mRNA test in cervical cytological samples of HC2-positive patients

The study aimed to assess the clinical utility in identifying CIN2 or worse (CIN2+), of the Pretect HPV-Proofer test for E6/E7 mRNA detection in Hybrid Capture 2 (HC2)-positive patients, who underwent colposcopy. In particular, the study analyzed the mRNA test performance as the third test in a subgroup of HC2+ patients with less severe than high-grade squamous intraepithelial lesions (HSIL-). We analyzed 464 cervico-vaginal samples by liquid-based cytology (LBC) and PreTect HPV-Proofer. Moreover 231 patients also had a biopsy at baseline and 75, with HSIL-, were followed up within 2 years by LBC, colposcopy, and histology when indicated. The highest sensitivity for CIN2+ belonged to the mRNA compared to LBC, at the HSIL+ threshold (72% vs. 58%), whereas the LBC showed the highest specificity and positive predictive value (PPV) (99 and 93% vs. 73 and 39%, respectively). Focusing on the 408 HSIL- patients, the mRNA positivity was significantly more associated with CIN2+ than CIN2- lesions (p < 0.0001). Moreover, among the 75 HSIL- followed up patients, the mRNA displayed high longitudinal Specificity (89%), even if the sensitivity and the PPV were low (50 and 20%, respectively). The present data suggest that the mRNA test may have a diagnostic and a potentially prognostic role in HC2+/HSIL- patients.     

First-line temozolomide chemotherapy in progressive low-grade astrocytomas after radiotherapy: molecular characteristics in relation to response

Only a few studies examined the effect of temozolomide (TMZ) in recurrent low-grade astrocytoma (LGA) after surgery, none of which included a homogeneous and sufficiently sized group of patients with progression after radiotherapy (RT). We evaluated a cohort of 58 patients treated with TMZ for progression after RT of a previous LGA and investigated the relation between outcome and mutations in the IDH1, IDH2, and TP53 genes, O⁶-methylguanine-methyltransferase (MGMT) promoter methylation, trisomy of chromosome 7, and loss of chromosomes 1p and 19q. All patients received first-line TMZ 200 mg/m²/day on days 1–5 every 4 weeks for a progressive LGA with a contrast-enhancing lesion on MRI after RT. Six months progression-free survival (PFS) was 67%, and the median overall survival was 14 months. An objective response was obtained in 54%. TP53 mutations and loss of chromosome 19q showed a borderline association with PFS, but none of the other molecular characteristics were correlated with the outcome to TMZ. Both a methylated MGMT promoter gene and IDH1 mutations were found in 86% of the tumor samples. A correlation was found between IDH1 mutations and MGMT promoter methylation (P < .001). Neither MGMT promoter methylation nor IDH1 mutations correlated with PFS, but the interval between the very first symptom of the LGA and the start of the TMZ was significantly longer in the patients with IDH1 mutations (P = .01) and a methylated MGMT promoter (P = .02). We conclude that MGMT promoter methylation and IDH1 mutations seem to predict survival from the time of diagnosis, but not PFS to TMZ.     

Receptor tyrosine kinases and TLR/IL1Rs unexpectedly activate myeloid cell PI3kγ, a single convergent point promoting tumor inflammation and progression

We show that imatinib, nilotinib, and dasatinib possess weak off-target activity against RAF and, therefore, drive paradoxical activation of BRAF and CRAF in a RAS-dependent manner. Critically, because RAS is activated by BCR-ABL, in drug-resistant chronic myeloid leukemia (CML) cells, RAS activity persists in the presence of these drugs, driving paradoxical activation of BRAF, CRAF, MEK, and ERK, and leading to an unexpected dependency on the pathway. Consequently, nilotinib synergizes with MEK inhibitors to kill drug-resistant CML cells and block tumor growth in mice. Thus, we show that imatinib, nilotinib, and dasatinib drive paradoxical RAF/MEK/ERK pathway activation and have uncovered a synthetic lethal interaction that can be used to kill drug-resistant CML cells in vitro and in vivo.