Rapid identification of Nocardia farcinica clinical isolates by a PCR assay targeting a 314-base-pair species-specific DNA fragment

Nocardia farcinica is the most clinically significant species within the Nocardia asteroides complex. Differentiation of N. farcinica from other members of N. asteroides complex is important because this species characteristically demonstrates resistance to several extended-spectrum antimicrobial agents. Traditional phenotypic characterization of this species is time- and labor-intensive and often leads to misidentification in the clinical microbiology laboratory. We previously observed a 409-bp product for all strains of N. farcinica by using randomly amplified polymorphic DNA analysis with the primer DKU49. In this investigation, the 409-bp fragment was sequenced and then used to design a specific primer pair, Nf1 (16-mer) and Nf2 (16-mer), complementary to the 409-bp fragment. PCR amplification of genomic DNA from 28 N. farcinica isolates with Nf1 and Nf2 generated a single intense 314-bp fragment. The specificity of the assay with these primers was verified, since there were no PCR amplification products observed from heterologous nocardial species (n = 59) or other related bacterial genera (n = 41). Restriction enzyme digestion using CfoI and direct sequencing of the 314-bp fragment further confirmed the specificity of the assay for N. farcinica. This highly sensitive and specific PCR assay provides a rapid (within 1 day of obtaining DNA) method for identification of this medically important emerging pathogen. Rapid diagnosis of N. farcinica infection may allow for earlier initiation of effective therapy, thus improving patient outcome.     

In vitro cultivation of microsporidia of clinical importance

Although attempts to develop methods for the in vitro cultivation of microsporidia began as early as 1937, the interest in the culture of these organisms was confined mostly to microsporidia that infect insects. The successful cultivation in 1969 of Encephalitozoon cuniculi, a microsporidium of mammalian origin, and the subsequent identification of these organisms as agents of human disease heightened interest in the cultivation of microsporidia. I describe the methodology as well as the cell lines, the culture media, and culture conditions used in the in vitro culture of microsporidia such as Brachiola (Nosema) algerae, Encephalitozoon cuniculi, E. hellem, E. intestinalis, Enterocytozoon bieneusi, Trachipleistophora hominis, and Vittaforma corneae that cause human disease.     

Free-living, Amphizoic and Opportunistic Amebas

Amebas belonging to the genera Naegleria, Acanthamoeba and Balamuthia are free-living, amphizoic and opportunistic protozoa that are ubiquitous in nature. These amebas are found in soil, water and air samples from all over the world. Human infection due to these amebas involving brain, skin, lung and eyes has increased significantly during the last 10 years. The epidemiology, immunology, protozoology, pathology, and clinical features of the infections produced by these protozoa differ strikingly. Infection by the pathogenic Naegleria fowleri is acquired by exposure to polluted water in ponds, swimming pools and man-made lakes. Raised temperatures during the hot summer months or warm water from power plants facilitate the growth of N. fowleri. N. fowleri is a thermophilic ameba that grows well in tropical and subtropical climates. The CNS infection, called Primary Amebic Meningoencephalitis (PAM), produced by N. fowleri is characterized by an acute fulminant meningoencephalitis leading to death 3–7 days after exposure. Victims are healthy, young individuals with a history of recent water-related sport activities. The portal of entry is the olfactory neuroepithelium. The pathologic changes are an acute hemorrhagic necrotizing meningoencephalitis with modest purulent exudate, mainly at the base of the brain, brainstem and cerebellum. Trophozoites can be seen within the CNS lesions located mainly around blood vessels. Thus far 179 cases have been reported; 81 in the USA alone. Balamuthia mandrillaris and several species of Acanthamoeba are pathogenic “opportunistic” free-living amebas which cause Granulomatous Amebic Encephalitis (GAE) in humans and animals. GAE is an infection, usually seen in debilitated, malnourished individuals, in patients undergoing immunosuppressive therapy for organ transplants and in Acquired Immunodeficiency Syndrome (AIDS). The granulomatous component is negligible, particularly in immunocompromised individuals. Pathologically these amebas produce a patchy, chronic or subacute granulomatous encephalitis with the presence of trophozoites and cysts. The portal of entry is probably through the respiratory tract or an ulceration of the skin reaching the CNS by hematogenous spread. As of October 1, 1996, 166 cases 1103 due to Acanthamoeba and 63 due to Balamothial of GAE have been reported from around the world. Of these 103 cases due to Acanthamoeba (72 have been reported in the USA alone, > 50 in AIDS). It is well known that several species of Acanthamoeba can also produce, chronic sight threatening ulceration of the cornea called Acanthamoeba keratitis (AK), mostly in contact lens wearers or in individuals with minor corneal abrasions. Hundreds of cases of AK have been documented world wide.     

Species-specific identification of microsporidia in stool and intestinal biopsy specimens by the polymerase chain reaction

In view of the increasing number of cases of human microsporidiosis, simple and rapid methods for clear identification of microsporidian parasites to the species level are required. In the present study, the polymerase chain reaction (PCR) was used for speciesspecific detection ofEncephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon (Septata) intestinalis, andEnterocytozoon bieneusi in both tissue and stool. Using stool specimens and intestinal biopsies of patients infected withEnterocytozoon bieneusi (n=9),Encephalitozoon spp. (n=2), andEncephalitozoon intestinalis (n=1) as well as stool spiked with spores ofEncephalitozoon cuniculi andEncephalitozoon hellem and tissue cultures ofEncephalitozoon cuniculi andEncephalitozoon hellem, three procedures were developed to produce PCR-ready DNA directly from the samples. Specific detection of microsporidian pathogens was achieved in the first PCR. The subsequent nested PCR permitted species determination and verified the first PCR products. Without exception, the PCR assay confirmed electron microscopic detection ofEnterocytozoon bieneusi andEncephalitozoon intestinalis in stool specimens and their corresponding biopsies and in spiked stool samples and tissue cultures infected withEncephalitozoon cuniculi andEncephalitozoon hellem. Moreover, identification ofEncephalitozoon spp. could be specified asEncephalitozoon intestinalis. Whereas standard methods such as light and transmission electron microscopy may lack sensitivity or require more time and special equipment, the PCR procedure described facilitates speciesspecific identification of microsporidian parasites in stool, biopsies, and, probably, other samples in about five hours.     

Pathology and pathogenesis of bioterrorism-related inhalational anthrax

During October and November 2001, public health authorities investigated 11 patients with inhalational anthrax related to a bioterrorism attack in the United States. Formalin-fixed samples from 8 patients were available for pathological and immunohistochemical (IHC) study using monoclonal antibodies against the Bacillus anthracis cell wall and capsule. Prominent serosanguinous pleural effusions and hemorrhagic mediastinitis were found in 5 patients who died. Pulmonary infiltrates seen on chest radiographs corresponded to intraalveolar edema and hyaline membranes. IHC assays demonstrated abundant intra- and extracellular bacilli, bacillary fragments, and granular antigen-staining in mediastinal lymph nodes, surrounding soft tissues, and pleura. IHC staining in lung, liver, spleen, and intestine was present primarily inside blood vessels and sinusoids. Gram's staining of tissues was not consistently positive. In 3 surviving patients, IHC of pleural samples demonstrated abundant granular antigen-staining and rare bacilli while transbronchial biopsies showed granular antigen-staining in interstitial cells. In surviving patients, bacilli were not observed with gram's stains. Pathological and IHC studies of patients who died of bioterrorism-related inhalational anthrax confirmed the route of infection. IHC was indispensable for diagnosis of surviving anthrax cases. The presence of B. anthracis antigens in the pleurae could explain the prominent and persistent hemorrhagic pleural effusions.     

Immunohistochemical, in situ hybridization, and ultrastructural localization of SARS-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in Taiwan

This article describes the pathological studies of fatal severe acute respiratory syndrome (SARS) in a 73-year-old man during an outbreak of SARS in Taiwan, 2003. Eight days before onset of symptoms, he visited a municipal hospital that was later identified as the epicenter of a large outbreak of SARS. On admission to National Taiwan University Hospital in Taipei, the patient experienced chest tightness, progressive dyspnea, and low-grade fever. His condition rapidly deteriorated with increasing respiratory difficulty, and he died 7 days after admission. The most prominent histopathologic finding was diffuse alveolar damage of the lung. Immunohistochemical and in situ hybridization assays demonstrated evidence of SARS-associated coronavirus (SARS-CoV) infection in various respiratory epithelial cells, predominantly type II pneumocytes, and in alveolar macrophages in the lung. Electron microscopic examination also revealed coronavirus particles in the pneumocytes, and their identity was confirmed as SARS-CoV by immunogold labeling electron microscopy. This report is the first to describe the cellular localization of SARS-CoV in human lung tissue by using a combination of immunohistochemistry, double-stain immunohistochemistry, in situ hybridization, electron microscopy, and immunogold labeling electron microscopy. These techniques represent valuable laboratory diagnostic modalities and provide insights into the pathogenesis of this emerging infection.     

Quantitation of Ergosterol Content: Novel Method for Determination of Fluconazole Susceptibility of Candida albicans

MIC end points for the most commonly prescribed azole antifungal drug, fluconazole, can be difficult to determine because its fungistatic nature can lead to excessive "trailing" of growth during susceptibility testing by National Committee for Clinical Laboratory Standards broth macrodilution and microdilution methods. To overcome this ambiguity, and because fluconazole acts by inhibiting ergosterol biosynthesis, we developed a novel method to differentiate fluconazole-susceptible from fluconazole-resistant isolates by quantitating ergosterol production in cells grown in 0, 1, 4, 16, or 64 microg of fluconazole per ml. Ergosterol was isolated from whole yeast cells by saponification, followed by extraction of nonsaponifiable lipids with heptane. Ergosterol was identified by its unique spectrophotometric absorbance profile between 240 and 300 nm. We used this sterol quantitation method (SQM) to test 38 isolates with broth microdilution end points of /=64 microg/ml (resistant) and 10 isolates with trailing end points by the broth microdilution method. No significant differences in mean ergosterol content were observed between any of the isolates grown in the absence of fluconazole. However, 18 susceptible isolates showed a mean reduction in ergosterol content of 72% after exposure to 1 microg of fluconazole/ml, an 84% reduction after exposure to 4 microg/ml, and 95 and 100% reductions after exposure to 16 and 64 microg of fluconazole/ml, respectively. Ten SDD isolates showed mean ergosterol reductions of 38, 57, 73, and 99% after exposure to 1, 4, 16, and 64 microg of fluconazole/ml, respectively. In contrast, 10 resistant isolates showed mean reductions in ergosterol content of only 25, 38, 53, and 84% after exposure to the same concentrations of fluconazole. The MIC of fluconazole, by using the SQM, was defined as the lowest concentration of the drug which resulted in 80% or greater inhibition of overall mean ergosterol biosynthesis compared to that in the drug-free control. Of 38 isolates which gave clear end points by the broth microdilution method, the SQM MIC was within 2 dilutions of the broth microdilution MIC for 33 (87%). The SQM also discriminated between resistant and highly resistant isolates and was particularly useful for discerning the fluconazole susceptibilities of 10 additional isolates which gave equivocal end points by the broth microdilution method due to trailing growth. In contrast to the broth microdilution method, the SQM determined trailing isolates to be susceptible rather than resistant, indicating that the SQM may predict clinical outcome more accurately. The SQM may provide a means to enhance current methods of fluconazole susceptibility testing and may provide a better correlation of in vitro with in vivo results, particularly for isolates with trailing end points.     

Prolonged Replication of a Type 1 Vaccine-Derived Poliovirus in an Immunodeficient Patient

VP1 sequences were determined for poliovirus type 1 isolates obtained over a 189-day period from a poliomyelitis patient with common variable immunodeficiency syndrome (a defect in antibody formation). The isolate from the first sample, taken 11 days after onset of paralysis, contained two poliovirus populations, differing from the Sabin 1 vaccine strain by ~10%, differing from diverse type 1 wild polioviruses by 19 to 24%, and differing from each other by 5.5% of nucleotides. Specimens taken after day 11 appeared to contain only one major poliovirus population. Evolution of VP1 sequences at synonymous third-codon positions occurred at an overall rate of ~3.4% per year over the 189-day period. Assuming this rate to be constant throughout the period of infection, the infection was calculated to have started ~9.3 years earlier. This estimate is about the time (6.9 years earlier) the patient received his last oral poliovirus vaccine dose, approximately 2 years before the diagnosis of immunodeficiency. These findings may have important implications for the strategy to eliminate poliovirus immunization after global polio eradication.     

Use of a repetitive DNA probe to type clinical and environmental isolates of Aspergillus flavus from a cluster of cutaneous infections in a neonatal intensive care unit

Aspergillus flavus is second to A. fumigatus as a cause of invasive aspergillosis, but no standard method exists for molecular typing of strains from human sources. A repetitive DNA sequence cloned from A. flavus and subcloned into a pUC19 vector, pAF28, was used to type 18 isolates from diverse clinical, environmental, and geographic sources. The restriction fragment length polymorphisms generated with EcoRI- or PstI-digested genomic DNA and probed with digoxigenin-labeled pAF28 revealed complete concordance between patterns. Eighteen distinct fingerprints were observed. The probe was used to investigate two cases of cutaneous A. flavus infection in low-birth-weight infants in a neonatal intensive care unit (NICU). Both infants were transported by the same ambulance and crew to the NICU on the same day. A. flavus strains of the same genotype were isolated from both infants, from a roll of tape used to fasten their umbilical catheters, from a canvas bag used to store the tape in the ambulance, and from the tape tray in the ambulance isolette. These cases highlight the need to consider exposures in critically ill neonates that might occur during their transport to the NICU and for stringent infection control practices. The hybridization profiles of strains from a second cluster of invasive A. flavus infections in two pediatric hematology-oncology patients revealed a genotype common to strains from a definite case patient and a health care worker. A probable case patient was infected with a strain with a genotype different from that of the strain from the definite case patient but highly related to that of an environmental isolate. The high degree of discrimination and reproducibility obtained with the pAF28 probe underscores its utility for typing clinical and environmental isolates of A. flavus.