Rapid method for detecting and differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in sputum by fluorescence in situ hybridization with DNA probes.

OBJECTIVE: In resource-limited tuberculosis-endemic countries, Mycobacterium tuberculosis in sputum is mainly detected by acid-fast bacillus (AFB) staining and the identification of sputum-derived cultures. PCR techniques are practical only in well-resourced laboratories. This study investigated the application of a rapid, simple, and inexpensive fluorescence in situ hybridization (FISH) assay to identify and differentiate M. tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM) in sputum. METHODS: The Mycobacterium/Nocardia Genus (MN Genus)-MTBC FISH assay performed in this study utilizes two different DNA probes labeled with different fluorescent molecules that hybridize respectively with 16S rRNA of the genus Mycobacterium and 23S rRNA of MTBC. The assay was tested on 202 patient sputum samples in Mangaluru, Karnataka State, India. Sputa were first liquefied and bacteria concentrated before performing the FISH assay and parallel culturing and AFB staining. The identities of cultured bacteria from DNA sequencing were compared with FISH assay findings from corresponding sputa. RESULTS: Of the 202 sputum samples tested, 67 reacted with both MN Genus-specific and MTBC-specific probes, none reacted only with the MTBC-specific probe, and 22 reacted only with the MN Genus-specific probe. The FISH assay yielded results in 2h and had a limit of detection of 2.2×104CFU/ml in sputum spiked with cultured M. tuberculosis. The diagnostic sensitivity, specificity, and positive and negative predictive values of the FISH assay for MTBC in patient sputa were 89.7%, 95.5%, 88.0%, and 92.6%, respectively. NTM were a significant cause of tuberculosis-like infections in Mangaluru. CONCLUSIONS: The MN Genus-MTBC dual probe fluorescence FISH assay previously applied to cultures can also be utilized in resource-limited tuberculosis-endemic countries for rapidly identifying and differentiating MTBC and NTM in sputum samples.

Dual color fluorescence in situ hybridization (FISH) assays for detecting Mycobacterium tuberculosis and Mycobacterium avium complexes and related pathogens in cultures.

Two rapid dual color fluorescence in situ hybridization (FISH) assays were evaluated for detecting M. tuberculosis and related pathogens in cultures. The MN Genus-MTBC FISH assay uses an orange fluorescent probe specific for the Mycobacterium tuberculosis complex (MTBC) and a green fluorescent probe specific for the Mycobacterium and Nocardia genera (MN Genus) to detect and distinguish MTBC from other Mycobacteria and Nocardia. A complementary MTBC-MAC FISH assay uses green and orange fluorescent probes specific for the MTBC and M. avium complex (MAC) respectively to identify and differentiate the two species complexes. The assays are performed on acid-fast staining bacteria from liquid or solid cultures in less than two hours. Forty-three of 44 reference mycobacterial isolates were correctly identified by the MN Genus-specific probe as Mycobacterium species, with six of these correctly identified as MTBC with the MTBC-specific probe and 14 correctly as MAC by the MAC-specific probe. Of the 25 reference isolates of clinically relevant pathogens of other genera tested, only four isolates representing two species of Corynebacterium gave a positive signal with the MN Genus probe. None of these 25 isolates were detected by the MTBC and MAC specific probes. A total of 248 cultures of clinical mycobacterial isolates originating in India, Peru and the USA were also tested by FISH assays. DNA sequence of a part of the 23S ribosomal RNA gene amplified by PCR was obtained from 243 of the 248 clinical isolates. All 243 were confirmed by DNA sequencing as Mycobacterium species, with 157 and 50 of these identified as belonging to the MTBC and the MAC, respectively. The accuracy of the MN Genus-, MTBC-and MAC -specific probes in identifying these 243 cultures in relation to their DNA sequence-based identification was 100%. All ten isolates of Nocardia, (three reference strains and seven clinical isolates) tested were detected by the MN Genus-specific probe but not the MTBC- or MAC-specific probes. The limit of detection for M. tuberculosis was determined to be 5.1x104 cfu per ml and for M. avium 1.5x104 cfu per ml in liquid cultures with the respective MTBC- and MAC-specific probes in both the MN Genus-MTBC and MTBC-MAC FISH assays. The only specialized equipment needed for the FISH assays is a standard light microscope fitted with a LED light source and appropriate filters. The two FISH assays meet an important diagnostic need in peripheral laboratories of resource-limited tuberculosis-endemic countries.

Fluorescence In Situ Hybridization (FISH) Assays for Diagnosing Malaria in Endemic Areas.

Malaria is a responsible for approximately 600 thousand deaths worldwide every year. Appropriate and timely treatment of malaria can prevent deaths but is dependent on accurate and rapid diagnosis of the infection. Currently, microscopic examination of the Giemsa stained blood smears is the method of choice for diagnosing malaria. Although it has limited sensitivity and specificity in field conditions, it still remains the gold standard for the diagnosis of malaria. Here, we report the development of a fluorescence in situ hybridization (FISH) based method for detecting malaria infection in blood smears and describe the use of an LED light source that makes the method suitable for use in resource-limited malaria endemic countries. The Plasmodium Genus (P-Genus) FISH assay has a Plasmodium genus specific probe that detects all five species of Plasmodium known to cause the disease in humans. The P. falciparum (PF) FISH assay and P. vivax (PV) FISH assay detect and differentiate between P. falciparum and P. vivax respectively from other Plasmodium species. The FISH assays are more sensitive than Giemsa. The sensitivities of P-Genus, PF and PV FISH assays were found to be 98.2%, 94.5% and 98.3%, respectively compared to 89.9%, 83.3% and 87.9% for the detection of Plasmodium, P. falciparum and P. vivax by Giemsa staining respectively.

K252a, a high-affinity nerve growth factor receptor blocker, improves psoriasis: an in vivo study using the severe combined immunodeficient mouse-human skin model.

The peripheral nervous system, in addition to its sensory and motor functions, can induce a local inflammatory response known as neurogenic inflammation. This phenomenon plays a critical role in several inflammatory diseases, e.g., asthma, atopy, rheumatoid arthritis, psoriasis, and ulcerative colitis. Neurogenic inflammation and the role of nerve growth factor (NGF) have been extensively studied in psoriasis. There are increased levels of NGF in the keratinocytes and upregulation of NGF receptor (NGF-R) in the cutaneous nerves of psoriatic plaques. NGF can influence all the salient pathologic events noticed in psoriasis such as proliferation of keratinocytes, angiogenesis, T cell activation, expression of adhesion molecules, proliferation of cutaneous nerves, and upregulation of neuropeptides. In this double-blinded, placebo-controlled study, we addressed the role of NGF/NGF-R in psoriasis in an in vivo system using the severe combined immunodeficient (SCID) mouse-human skin model of psoriasis. The transplanted psoriatic plaques on the SCID mice (n=12) were treated with K252a, a high-affinity NGF receptor blocker. Psoriasis significantly improved following 2 wk of therapy. The length of the rete pegs changed from 308.57+/-98.72 to 164.64+/-46.78 microm (p<0.01, Student's t test). A similar improvement of psoriasis was observed by directly inhibiting NGF with NGF-neutralizing antibody. NGF-neutralizing antibody in normal saline at 10 ng (n=4) and 20 ng (n=4) per kilogram of body weight doses were used. Both doses of NGF-neutralizing antibody reduced rete peg lengths significantly, e.g., from 298.5+/-42.69 to 150.52+/-32.93 microm (p<0.05, Student's t test). This study provides evidence for the role of NGF and its high-affinity receptor in the pathogenesis of psoriasis and insights to develop novel therapeutic modalities.

High-throughput detection of submicroscopic deletions and methylation status at 15q11-q13 by a photo-cross-linking oligonucleotide hybridization assay.

BACKGROUND: Current technologies for assessing genetic deletions and duplications of greater than one kilobase are labor-intensive or rely on PCR-based methods, and none offers the ability to simultaneously detect dosage abnormalities, assess 5'-to-3' cytosine-guanosine (CpG) methylation, and interrogate single-nucleotide polymorphisms (SNPs). We describe a high-throughput platform for direct gene-dosage determination capable of concurrent assessment of other forms of gene modification. METHODS: We used a light-activated interstrand nucleic acid cross-linking system (XLnt technology) to determine gene dosage at the 15q11-q13 deletion/duplication locus. We incorporated restriction enzyme digestion of genomic DNA into the method to assess CpG methylation in parallel with gene dosage. For method validation we used DNA from 31 cell lines with previously characterized 15q11-q13 gene dosage and parental origin status. Diagnostic cutoffs were set at 0.5 +/- 0.15, 1 +/- 0.15-0.25, and 2 +/- 0.3. RESULTS: Dosage-only experiments showed discrimination of deletions (n = 21) from healthy controls (NCs; n = 27) in all samples. Five of 49 samples gave results outside of specification. Concurrent evaluation of dosage and CpG methylation yielded dosage results within specification for 18 of 19 deletion and 8 of 12 NC samples. Paternal deletion and NC methylation pattern results were within specification in 17 of 19 and 9 of 12 runs, respectively. No overlap was demonstrated between value sets for the two groups. CONCLUSIONS: The XLnt technology provides a rapid, high-throughput platform for the accurate determination of gene dosage. The flexibility of this technology allows parallel interrogation of gene dosage, CpG methylation, and SNPs.