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. 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. 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. 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.

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.

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.

We have amplified by PCR Pneumocystis carinii cytoplasmic small-subunit rRNA (variously referred to as 16S-like or 18S-like rRNA) genes from DNA extracted from bronchoalveolar lavage and induced sputum specimens from patients positive for P. carinii and from infected ferret lung tissue. The amplification products were cloned into pUC18, and individual clones were sequenced. Comparison of the determined sequences with each other and with published rat and partial human P.carinii small-subunit rRNA gene sequences reveals that, although all P. carinii small-subunit rRNAs are closely related (approximately 96% identity), small-subunit rRNA genes isolated from different host species (human, rat, and ferret) exhibit distinctive patterns of sequence variation. Two types of sequences were isolated from the infected ferret lung tissue, one as a predominant species and the other as a minor species. There was 96% identity between the two types. In situ hybridization of the infected ferret lung tissue with oligonucleotide probes specific for each type revealed that there were two distinct strains of P. carinii present in the ferret lung tissue. Unlike the ferret P. carinii isolates, the small-subunit rRNA gene sequences from different human P. carinii isolates have greater than 99% identity and are distinct from all rat and ferret sequences so far inspected or reported in the literature. Southern blot hybridization analysis of PCR amplification products from several additional bronchoalveolar lavage or induced sputum specimens from P. carinii-infected patients, using a 32P-labeled oligonucleotide probe specific for human P. carinii, also suggests that all of the human P. carinii isolates are identical. These findings indicate that human P. carinii isolates may represent a distinct species of P. carinii distinguishable from rat and ferret P. carinii on the basis of characterization of small-subunit rRNA gene sequences.

Background: With more than 300,000 new cases reported each year in the United States of America (USA), Lyme disease is a major public health concern. Borrelia burgdorferi sensu stricto (Bbss) is considered the primary agent of Lyme disease in North America. However, multiple genetically diverse Borrelia species encompassing the Borrelia burgdorferi sensu lato (Bbsl) complex and the Relapsing Fever Borrelia (RFB) group are capable of causing tickborne disease. We report preliminary results of a serological survey of previously undetected species of Bbsl and RFB in California and Mexico using a novel immunoblot technique. Methods: Serum samples were tested for seroreactivity to specific species of Bbsl and RFB using an immunoblot method based on recombinant Borrelia membrane proteins, as previously described. A sample was recorded as seropositive if it showed immunoglobulin M (IgM) and/or IgG reactivity with at least two proteins from a specific Borrelia species. Results: The patient cohort consisted of 90 patients residing in California or Mexico who met the clinical case definition of chronic Lyme disease. Immunoblot testing revealed that 42 patients were seropositive for Bbsl (Group 1), while 56 patients were seropositive for RFB (Group 2). Eight patients were seropositive for both Bbsl and RFB species. Group 1 included patients who were seropositive for Bbss (14), B. californiensis (eight), B. spielmanii (10), B. afzelii/B. garinii (10), and mixed infections that included B. mayonii (three). Group 2 included patients who were seropositive for B. hermsii (nine), B. miyamotoi (seven), B. turicatae (nine), and B. turcica (two). In the remaining Group 1 and Group 2 patients, the exact Borrelia species could not be identified using the immunoblot technique. Conclusions: Lyme disease is associated with a diverse group of Borrelia species in California and Mexico. Current testing for Lyme disease focuses on detection of Bbss, possibly resulting in missed diagnoses and failure to administer appropriate antibiotic therapy in a timely manner. The genetic diversity of Borrelia spirochetes must be considered in future Lyme disease test development.