Light microscopy provides a simple, cost-effective, and vital method for the diagnosis and screening of hematologic and infectious diseases. In many regions of the world, however, the required equipment is either unavailable or insufficiently portable, and operators may not possess adequate training to make full use of the images obtained. Counterintuitively, these same regions are often well served by mobile phone networks, suggesting the possibility of leveraging portable, camera-enabled mobile phones for diagnostic imaging and telemedicine. Toward this end we have built a mobile phone-mounted light microscope and demonstrated its potential for clinical use by imaging P. falciparum-infected and sickle red blood cells in brightfield and M. tuberculosis-infected sputum samples in fluorescence with LED excitation. In all cases resolution exceeded that necessary to detect blood cell and microorganism morphology, and with the tuberculosis samples we took further advantage of the digitized images to demonstrate automated bacillus counting via image analysis software. We expect such a telemedicine system for global healthcare via mobile phone – offering inexpensive brightfield and fluorescence microscopy integrated with automated image analysis – to provide an important tool for disease diagnosis and screening, particularly in the developing world and rural areas where laboratory facilities are scarce but mobile phone infrastructure is extensive.

/pdf/mobile-phone-based-clinical-microscopy-for-global-health-3siutbfblo.pdf

Mobile phone based clinical microscopy for global health applications.

Mycobacteria are the causative organisms for diseases such as tuberculosis (TB), leprosy, Buruli ulcer, and pulmonary nontuberculous mycobacterial disease, to name the most important ones. In 2015, globally, almost 10 million people developed TB, and almost half a million patients suffered from its multidrug-resistant form. In 2016, a total of 9,287 new TB cases were reported in the United States. In 2015, there were 174,608 new case of leprosy worldwide. India, Brazil, and Indonesia reported the most leprosy cases. In 2015, the World Health Organization reported 2,037 new cases of Buruli ulcer, with most cases being reported in Africa. Pulmonary nontuberculous mycobacterial disease is an emerging public health challenge. The U.S. National Institutes of Health reported an increase from 20 to 47 cases/100,000 persons (or 8.2% per year) of pulmonary nontuberculous mycobacterial disease among adults aged 65 years or older throughout the United States, with 181,037 national annual cases estimated in 2014. This review describes contemporary methods for the laboratory diagnosis of mycobacterial diseases. Furthermore, the review considers the ever-changing health care delivery system and stresses the laboratory's need to adjust and embrace molecular technologies to provide shorter turnaround times and a higher quality of care for the patients who we serve.

Practice Guidelines for Clinical Microbiology Laboratories: Mycobacteria

This chapter deals with Mycobacterium the only genus in the family Mycobacteriaceae and related to other mycolic acid-containing genera. The genus Mycobacterium includes obligate pathogens, opportunistic pathogens, and saprophytes. Ben Salah et al. found clinical M. avium complex (MAC) isolates that appear to represent three other new species: M. marseillense, M. timonense, and M. bouchedurhonense. Many different types of clinical specimens may be collected for mycobacteriological analyses. A majority of clinical specimens originate from the respiratory tract (sputum, tracheal and bronchial aspirates, and bronchoalveolar lavage specimens), but urine, gastric aspirates, tissues, biopsy specimens, and normally sterile body fluids such as cerebrospinal fluid and pleural and pericardial aspirates are other commonly submitted specimens. A majority of disseminated mycobacterial infections are due to MAC. Appropriate pretreatment and processing procedures (homogenization, decontamination, concentration, culture media, and conditions of incubation) must be selected to facilitate optimum recovery of mycobacteria. With the advent of molecular techniques designed for molecular epidemiology, cross-contamination either linked to laboratory procedures or, more rarely, to contaminated bronchoscopes can easily be proven. Laboratory aspects of cross-contamination are addressed in the chapter. Quality control (QC) is vital for monitoring a laboratory’s effectiveness in detecting and isolating mycobacteria. Accurate identification of nontuberculous mycobacteria (NTM) will prevent rarely encountered pathogens from being mistaken for nonpathogenic species.

Mycobacterium: General Characteristics, Laboratory Detection, and Staining Procedures*

Tuberculosis (TB) is the leading cause of death from an infectious bacterial disease. Poor diagnostic tools to detect active disease plague TB control programs and affect patient care. Accurate detection of live Mycobacterium tuberculosis (Mtb), the causative agent of TB, could improve TB diagnosis and patient treatment. We report that mycobacteria and other corynebacteria can be specifically detected with a fluorogenic trehalose analog. We designed a 4-N,N-dimethylamino-1,8-naphthalimide-conjugated trehalose (DMN-Tre) probe that undergoes >700-fold increase in fluorescence intensity when transitioned from aqueous to hydrophobic environments. This enhancement occurs upon metabolic conversion of DMN-Tre to trehalose monomycolate and incorporation into the mycomembrane of Actinobacteria. DMN-Tre labeling enabled the rapid, no-wash visualization of mycobacterial and corynebacterial species without nonspecific labeling of Gram-positive or Gram-negative bacteria. DMN-Tre labeling was detected within minutes and was inhibited by heat killing of mycobacteria. Furthermore, DMN-Tre labeling was reduced by treatment with TB drugs, unlike the clinically used auramine stain. Lastly, DMN-Tre labeled Mtb in TB-positive human sputum samples comparably to auramine staining, suggesting that this operationally simple method may be deployable for TB diagnosis.

/pdf/rapid-detection-of-mycobacterium-tuberculosis-in-sputum-with-2u9w3t6v2j.pdf

Rapid detection of Mycobacterium tuberculosis in sputum with a solvatochromic trehalose probe.

We conducted a multicenter study to determine whether Mycobacterium tuberculosis complex (MTBC) cultures in automated broth-based systems could reliably be considered negative sooner than 6 weeks. Laboratory sites used Bactec MGIT or BacT/Alert and tracked results of time to detection of all mycobacteria (TTD-all, n = 1547) and of MTBC (TTD-MTBC, n = 466) over 6-month periods from primarily (93%) respiratory specimens. Cumulative percentages by day detected and median TTD of initial and follow-up specimens were analyzed. The median TTD-MTBC for MGIT (n = 6 sites) was 14 days. For laboratories using standard processing procedures, 100% of MTBC were detected from initial and follow-up specimens in 28 and 35 days, respectively, and no yield of MTBC on solid or MGIT liquid media was observed after 5 weeks. The median TTD-MTBC for BacT/Alert (n = 3 sites) was 18 days, with 95% and 100% detected within 37 and 42 days, respectively. Analysis of TTD of positive MTBC cultures in broth can predict the probability of culture negativity at defined time points. Receipt of interim negative reports earlier than 6 weeks could assist clinicians in considering alternative diagnoses and could alter the timing and prioritization of public health interventions. Laboratories should analyze their own TTD data to inform protocol decisions. Laboratories using MGIT could issue reports of no growth of MTBC on initial specimens as early as 4 weeks and for patients undergoing treatment as early as 5 weeks postinoculation.

Probability of Negative Mycobacterium tuberculosis Complex Cultures Based on Time to Detection of Positive Cultures: a Multicenter Evaluation of Commercial-Broth-Based Culture Systems

Rapid detection of mycobacterial disease is essential. Using multiple specimen types and concentrations of mycobacteria, we compared two commercial auramine O stains. The more rapid stain permitted consistent acid-fast bacillus quantitation and exhibited less debris staining, and the staining procedure required less time (∼2 min) to perform. These results suggest that the rapid stain may be more cost-effective and efficient for use in clinical laboratories.

Evaluation of a Rapid Fluorescent Staining Method for Detection of Mycobacteria in Clinical Specimens

Differences in time to detection and recovery of Mycobacterium spp. between the MGIT 960 and the BacT/ALERT MB Automated Culture Systems

Two methods were compared for decreasing bacterial contamination in the BacT/Alert mycobacterial culture detection system. Two concentrations, 0.5 ml (standard amount) and 1.0 ml, of mycobacterial antibiotic supplement were evaluated. Contamination rates were 14% and 6% for the standard and the doubled concentrations, respectively. This difference was statistically significant (P < 0.005).

Methods for Reducing Bacterial Contamination in the BacT/Alert Mycobacterial Culture Detection System

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Evaluation of a Rapid Fluorescent Staining Method for Detection of Mycobacteria in Clinical Specimens

Differences in time to detection and recovery of Mycobacterium spp. between the MGIT 960 and the BacT/ALERT MB Automated Culture Systems

Methods for Reducing Bacterial Contamination in the BacT/Alert Mycobacterial Culture Detection System