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Genus Candida covers more than 50 species, half of which can cause infections in humans. Some of the Candida species exhibit drug resistance; therefore, there is an urgent need for rapid and accurate differentiation for rendering appropriate and effective management. Here, we report a new methodology employing real-time polymerase chain reaction (RTPCR) and melting temperature analyses (MTA) procedures. Fungal ribosomal internal transcribed spacer 2 (ITS2) has been confirmed with variable nucleotide sequences, which makes it possible to differentiate one species from another by checking their melting temperature following PCR amplification. The universal primers (panFg) covering entire ITS2 region, from 5.8S to 28S rRNA genes, were designed to differentially identify most Candida species with RTPCR-MTA procedure. Nucleic acids from five genomes of closely related Candida species, which were experimentally spiked into human blood, were extracted and amplified. PCR amplicons were called for melting temperature of Candida albicans (87.49°C), C. glabrata (86.85°C), C. krusei (90.24°C), C. parapsilosis (86.22°C), and C. tropicalis (86.08°C). The melting temperature of each amplicon was consistent and reproducible in three replicate experiments (SD ± 0.04-0.32). The new RTPCR-MTA methodology showed promise in differential diagnosis of closely related Candida species from environmental and clinical samples.

Brucellosis is a zoonotic infection transmitted from animals to human by ingestion of infected food products, direct contact with an infected animal, or inhalation of aerosols. Brucella infection-induced osteomyelitis may present only with nonspecific clinical and radiographic findings, mild elevations in serum inflammatory markers, as well as nonspecific histological changes. We studied a case of an Iraqi war veteran with multifocal vertebral body and left iliac bone lesions on radio nucleotide scans and magnetic resonance imaging, clinically suspected osteomyelitis possibly because of Brucella. Although histomorphological findings were nonspecific, consisting of chronic inflammatory cell infiltrate and reactive fibrosis, tissue gram and silver impregnation stains of bone biopsies were informative, revealing gram-negative coccobacilli consistent in size with Brucella species. Total nucleic acids were extracted from formalin-fixed paraffin-embedded tissues and amplified by sequential real-time polymerase chain reaction, targeting genes coding (1) outer membrane protein (omp-31) of Brucella species and (2) insertion sequence (IS711) of Brucella abortus (b-abt). Polymerase chain reaction results confirmed B. abortus as the causative pathogens for presumed diagnosis of Brucella osteomyelitis.

Burkholderia pseudomallei and B. mallei are two highly pathogenic bacteria responsible for melioidosis and glanders, respectively. Our laboratory developed hydrolysis probe-based real-time polymerase chain reaction assays targeting type three secretion system (TTS) and transposase family protein (TFP) of B. pseudomallei and B. malli, respectively. The assays were validated for target specificity, amplification sensitivity, and reproducibility. A bacterial DNA panel, composed of B. pseudomallei (13 strains), B. mallei (11 strains), Burkholderia species close neighbors (5 strains), and other bacterial species (17 strains), was prepared for specificity testing. Reference DNAs from B. pseudomallei and B. mallei bacterial cultures were used as controls for amplification, limit of detection, and reproducibility testing. The two TaqMan assays, Bp-TTS 1 and Bm-TFP, were optimized and applied in a retrospective study of archived cases from the Armed Forces Institute of Pathology. We tested 10 formalin-fixed paraffin-embedded blocks originally from autopsy specimens of patients who died of melioidosis or glanders during or after overseas tours in 1960s. Polymerase chain reaction results confirmed that DNA samples from formalin-fixed paraffin-embedded blocks of eight patients with melioidosis were positive for Bp-TTS 1 target and two patients with glanders were positive for Bm-TFP target.

Chronic persistent infections by mycoplasmas induced malignant transformation of C3H mouse embryo cells that normally had never been reported to undergo spontaneous transformation. This mycoplasma-mediated oncogenic process had a long latency (more than 7 weeks of continuous mycoplasmal infection) and showed a multistage progression characterized by reversibility (at least up to 11 weeks of mycoplasmal infection) and irreversibility of malignant properties upon removal of the mycoplasma from culture. Further prolonged infections (18 weeks) by Mycoplasma fermentansor M. penetransresulted in permanent transformation of these C3H cells that no longer required the continued presence of the transformation-inducing mycoplasmas in cultures to retain their malignant properties. Previous studies of viral oncogenesis revealed that virus-transformed cells always had viral gene(s) present. Integration of viral gene(s) apparently played an important role in the process of oncogenesis. In this study, we examined if the continued presence of any mycoplasmal gene(s) in mammalian cells, in whatever form, was also crucial in causing malignant cell transformation. Representational difference analysis (RDA) was a recently developed powerful technique to compare differences between two complex genomes. In the RDA system, subtractive and kinetic enrichment was used to purify and isolate restriction endonuclease gene f ragment(s) of mycoplasmal origin, presumably present only in mycoplasma-transformed C3H cells, but not in nonmycoplasma-exposed control C3H cells. After three rounds of subtractive hybridization following PCR enrichment for each of three different restriction enzymes DNA digests, no gene fragment of mycoplasmal origin was amplified or identified in the permanently transformed C3H cells. Differing from tumorigenesis in animal cells induced by most oncogenic viruses or in plant cells induced by Agrobacteria, mycoplasmas evidently did not cause malignant transformation by integrating their gene(s) into the mammalian cell genome.

C3H mouse embryo cells, which normally have low inherent spontaneous transformation, underwent malignant transformation while chronically infected with Mycoplasma fermentansor Mycoplasma penetrans.This mycoplasma-mediated oncogenic process had long latency (more than 7 weeks of persistent mycoplasmal infection) and showed multistage progression characterized by reversibility and irreversibility of malignant properties upon removal of M. fermentansfrom culture. Marked expression of H-rasand c-mycmRNA, but not N-myc, src, N-ras, or p53 mRNA, was found in the mycoplasma-transformed C3H cells that exhibited characteristic malignant properties of morphological changes and uncontrolled cell growth. However, at least up to the eleventh week of persistent mycoplasma infection, the marked expression of H-rasor c-mycmRNA in C3H cells depended on continued presence of the mycoplasma in culture. H-rasor c-mycmRNA rapidly declined to the undetectable low levels of nontransformed parental C3H cells, and all malignant properties of the once-fully-transformed C3H cells quickly reversed, if M. fermentanswas eradicated from culture. In comparison, infection with M. penetransfor 7 or 11 weeks also induced a high level of H-ras, but not c-myc, mRNA expression in C3H cells. Despite having prominent amount of steady-state H-rasmRNA, these M. penetrans—infected C3H cells did not show any sign of malignant transformation. Thus, marked expression of H-rasgene alone was not sufficient to effect transformation in C3H cells. Interestingly, after a further prolonged (18 weeks) infection with either M. fermentansor M. penetrans, C3H cells revealed prominent chromosomal changes, expressed constitutively (with or without the presence of the transforming mycoplasmas) at high levels of both H-rasand c-mycmRNA and became permanently transformed. These cells were able to form tumors in animals.

An investigation of human splenic LAK cells killing autologous tumor invivohas been performed in this study. Briefly, an ovary embryo carcinoma (OEC) removed surgically from patient was transplanted into nude mice (s. c.) and culturedin vitro, that would be used as targets. Lymphokine-activated killer (LAK) cells were generated from splenic lymphocytes of the OEC patient, who died two and half months after operation, by co- culture with recombinant human interleukin- 2 (rIL- 2) invitro. The results from Winn’ s test in nude mice suggested that these LAK cells could effectively inhibit the tumorigenicity of autologous tumorin vivo.