Diagnosis of Latent Mycobacterium tuberculosis Infection in the Era of Interferon Gamma Release Assays

Mycobacterium tuberculosis (MTB), an acid-fast bacterium, is a leading cause of respiratory illness and death worldwide. Individuals can be latently infected and can harbor the organism without clinical evidence of disease for years. Screening and treatment of numerous populations, including health care workers, recent contacts of actively infected individuals, immunocompromised individuals, children, and immigrants from countries where MTB is endemic, is essential to eradicate the infection by 2050, as the World Health Organization envisions. Detection of active and latent infection historically has utilized tuberculin skin tests and other clinical findings. Interferon gamma release assays can test whether a patient has had MTB exposure with improved sensitivity and specificity over tuberculin skin testing. This article reviews the history of MTB testing, compares available interferon gamma release assays, and discusses the new developments related to latent-MTB infection testing.     

Wohlfahrtiimonas chitiniclastica: Two Clinical Cases and a Review of the Literature

Wohlfahrtiimonas chitiniclastica has been identified as an emerging pathogen predominantly associated with cutaneous myiasis and poor hygiene. Traditional biochemical methods can be unreliable and misleading when identifying this organism, and modern molecular techniques are often necessary for high-confidence identification. There have been very few cases of human infection with W. chitiniclastica reported in the literature. We present two recent cases identified at the University of Kentucky Medical Center (a male with myiasis related to gangrene and an elderly female with a left-leg wound and myiasis), both of which were identified using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for identification, and describe the clinical and microbiologic characteristics associated with this microorganism.     

Amplification and abnormal chromosomal distribution of ribosomal genes (rDNA) in rat erythroleukemia cells

The ribosomal cistrons (rDNA)/genome ratio was measured in five cell lines derived from three chemically induced erythroblastic leukemias (D-1, D-2, and NE26) in the Long-Evans (LE) rat and compared with values in the normal liver, bone marrow, and fetus. The ratio was 20–42% higher in the leukemias than in normal tissues. The number of autoradiographic silver grains of ¹²⁵I-labeled rRNA hybridized in situ over three nucleolus organizer regions (NORs) of leukemia cells was determined and compared with that of the normal cells. Although the distribution of silver grains of normal cells averaged 44.6%, 25.9%, and 29.5% in NORs of chromosomes #3, #11, and #12, respectively, their distribution was abnormal in two of the leukemias examined; rDNA was amplified in chromosomes #12 of two sublines (K1DA and K1DB) of one leukemis (D-1), and in one chromosome #3 of two sublines (K2D and K3D) of another leukemia (D-2). We consider the possibility that these abnormal patterns of rDNA distribution are related to the increase in rDNA in leukemia cells.     

Phenotypic Methods for Detection of Carbapenemase Production in Carbapenem-Resistant Organisms: What Method Should Your Laboratory Choose?

Antimicrobial resistance is a rising problem among Gram-negative organisms, and resistance to carbapenems is a special concern and an urgent public health threat. Rapid detection of carbapenem resistance, and carbapenemase production specifically, is becoming increasingly important for guiding infection control strategies and for therapeutic management of patients. Several types of phenotypic tests for the detection of carbapenemase production continue to be developed and include culture/growth-based methods (carbapenem inactivation method [CIM]/modified CIM [mCIM]), colorimetric assays (Carba NP and derivatives), matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based tests, and lateral-flow assays. Here, we describe the tests currently available, their performance characteristics, and how to select and verify/validate a test for the specific needs of a laboratory.     

Come in Out of the Cold: Alternatives to Freezing for Microbial Biorepositories

Biorepositories are “libraries” in which biospecimens, bacteria, or DNA and RNA extracts are stored for either clinical or research purposes. Such specimens enable modern molecular-based research and could support method verification, validation, quality control, and, in some cases, proficiency testing in clinical laboratories. Cryopreservation of extracted nucleic acids ensures the stability and longevity of DNA and RNA from patient samples, with the most common methods used for long-term storage of samples being the use of ?80°C freezers or liquid nitrogen. Frozen biospecimens are crucial for translational research as they contain well-preserved nucleic acids and protein; however, traditional ?80°C freezers consume both energy and space, with costs of maintenance and repairs reaching thousands of dollars annually to freeze and protect biospecimens. Additionally, liquid nitrogen is hazardous to work with, and failure to maintain adequate levels in storage containers can result in loss of specimens. Recently, new room temperature, or “green,” technology has been developed for dry storage of nucleic acids, ultimately reducing costs in terms of energy output and carbon footprint. This review compares and contrasts the use of dry-storage infrastructure with that of freezing samples, in terms of its use in clinical microbiology and highlights considerations to be made if implementing the technologies. Storage alternatives to freezers that equal or exceed their performance with regard to sample preservation and protection against degradation while at the same time reducing space requirements, costs, and energy consumption could be a financial and operational benefit if properly deployed and characterized. Perhaps it is time for laboratories to consider getting molecular quality control material and remnant extracted samples in from the cold and to evaluate the use of dry, room temperature storage technology for use in our microbial biorepositories.     

The relative merits of cromolyn sodium and high-dose theophylline therapy in childhood asthma

The use of cromolyn sodium (SCG) and high-dose theophylline (HDT) in the treatment of chronic perennial asthma in children is reviewed. It is noted that the regimens are only suitable for children with persistent symptoms uncontrolled by simpler forms of treatment. The methods of administration and dosage based on pharmacologic data are considered, and the potential importance of long-acting theophylline and nebulized cromolyn preparations is noted. Short-term studies have confirmed the efficacy of both drugs, and a comparative study showed little difference between them. Long-term studies of SCG have demonstrated its value to some 66% of children without serious side effects. No formal long-term studies have been carried out on HDT. Side effects from theophylline can often be eliminated by careful control of blood levels. From published evidence, neither SCG nor HDT is effective in steroid-dependent asthmatic children, and they contribute little, if anything, to management in such cases. The difference in cost of the drugs is small when all factors are considered, and either regimen is justified by the saving in medical expenses when used for carefully selected patients.     

MicroRNA155 Plays a Critical Role in the Pathogenesis of Cutaneous Leishmania major Infection by Promoting a Th2 Response and Attenuating Dendritic Cell Activity

Interferon (IFN)-γ is indispensable in the resolution of cutaneous leishmaniasis (CL), while the Th2 cytokines IL-4, IL-10, and IL-13 mediate susceptibility. A recent study found that miR155, which promotes CD4⁺ Th1 response and IFN-γ production, is dispensable in the control of Leishmania donovani infection. Here, the role of miR155 in CL caused by L. major was investigated using miR155-deficient (miR155^−/−) mice. Infection was controlled significantly quicker in the miR155^−/− mice than in their wild-type (WT) counterparts, indicating that miR155 contributes to the pathogenesis of CL. Faster resolution of infection in miR155^−/− mice was associated with increased levels of Th1-associated IL-12 and IFN-γ and reduced production of Th2- associated IL-4, IL-10, and IL-13. Concentrations of IFN-γ⁺CD8⁺ T cells and natural killer cells in draining lymph nodes were significantly higher in the L. major−infected miR155^−/− mice than in the infected WT mice, as indicated by flow-cytometry. After in vitro IFN-γ stimulation, nitric oxide and IL-12 production were increased, IL-10 production was decreased, and parasite clearance was enhanced in L. major−infected miR155^−/− DCs compared to those in WT DCs. Furthermore, IFN-γ production from activated miR155^−/− T cells was significantly enhanced in L. major−infected miR155^−/− DCs. Together, these findings demonstrate that miR155 promotes susceptibility to CL caused by L. major by promoting Th2 response and inhibiting DC function.

Leishmania are obligate intracellular protozoans that infect phagocytes and cause a spectrum of clinical diseases such as cutaneous leishmaniasis (CL) and visceral leishmaniasis. Common in the tropical and subtropical regions, leishmaniasis affects over 1 billion people worldwide, with an incidence of up to 1 million cases per year.¹ CL is the most common type of Leishmania infection, manifesting as localized skin lesions that can become chronic, leading to significant tissue destruction and disfigurement.^2,3 It is well documented that the induction of a Th1 response and interferon (IFN)-γ are indispensable in the resolution of CL caused by Leishmania major,⁴ whereas disease progression is associated with the induction of a Th2 response and the production of cytokines such as IL-4 and IL-10.⁵ Establishing a disease-resolving response in the host is largely dependent on the ability to mount an appropriate Th1 immune response.⁴ Crucial in this response is the stimulation and activation of DCs that direct T-cell proliferation and differentiation toward IFN-γ–producing Th1 cells.^6,7 In addition to activating of phagocytic cells, IFN-γ induces the production of reactive nitrogen species, specifically nitric oxide (NO), leading to enhanced parasite clearance.⁴miR155 is a recognized regulator of immune cell function and immune response. miR155 enhances macrophage and DC activation and induces inflammatory response,^8,9 and up-regulation of miR155 in CD4⁺ T cells promotes preferential Th1 differentiation and IFN-γ production¹⁰ by suppressing the expression of suppressor of cytokine signaling (SOCS)-1.11, 12, 13, 14 Conversely, miR155 gene–deficient mice exhibit diminished levels of Th1/Th17 cells, macrophages, and DCs.¹⁵ miR155 has also been shown to play a role in regulating effector Th2 response.16, 17, 18 Collectively, these findings suggest that miR155 regulates both Th1 and Th2 responses, which control the outcome of CL caused by L. major. Therefore, the role of miR155 in immunity to L. major using miR155^−/− mice was investigated in the present study. The findings show that miR155 is not required for the induction of a Th1 response and IFN-γ in L. major infection. Rather, miR155 plays a disease-exacerbating role in CL by attenuating DC function and Th1 response and promoting Th2 response.