BACKGROUND AND OBJECTIVE

Nontuberculous mycobacteria (NTM) lung disease appears like tuberculosis infection but is resistant to primary anti-tuberculosis drugs. Hence, patients whose sputum sample tests positive for acid-fast bacilli (AFB) and bacterial culture for several times should be assessed for colonization or infection with NTM in a damaged lung secondary to TB. In such cases, though drug-resistant TB may be adequately treated, treatment may need to be directed towards the NTM as well. In NTM therapy, the duration and choice of treatment agent is based upon the specific organism and disease extent. This study used one-step multiplex PCR (mPCR) assay for rapid differentiation of solid cultures in Ogawa medium as Mycobacterium tuberculosis (MTB) and/or NTM.

METHODS

A total of 80 stocked isolates obtained from the Lung Center of the Philippines from January to December 2018 were screened for NTM in terms of growth in Ogawa medium, acid fastness, and MPT64 TB antigen test result. These were from sputum specimens of multidrug-resistant tuberculosis (MDR-TB) patients. DNA was extracted from cultures (n=55) grown in Ogawa medium and one-step mPCR was performed to identify NTM to the species level.

RESULTS

Out of 80 samples screened, a total of 55 isolates were identified as NTM. One-step mPCR identified 12.73% (7/55) as M. abscessus, 34.55% (19/55) as M. massiliense, 1.82% (1/55) as M. kansasii, and 50.91% (28/55) were identified only up to genus Mycobacteria spp. Neither M. avium complex nor M. intracellulare was identified among the samples tested.

CONCLUSION

One-step mPCR was able to identify isolates as MTB or NTM coinciding with the initial screening using MPT64 TB antigen test. Multiplex PCR has given a more specific identificati on to the species level. The use of mPCR in identifying MTB and clinically significant NTM’s is suitable for the adequate treatment of mycobacterial infection.

Human ; Bacteria ; Multiplex Pcr ; Multiplex Polymerase Chain Reaction ; Mycobacteria ; Mycobacterium ; Tuberculosis, Multidrug-resistant

Background and Objectives: The hospital as health care facility has also become a source of infection that provides a place for different microbiological agents such as fungi. Exposure to these organisms is specifically detrimental to highly immunocompromised in-house patients. This study aimed to 1) detect the presence of fungi in a public tertiary hospital in Metro Manila; 2) determine the dominating fungal organism; and 3) describe the environmental conditions and physical factors affecting the proliferation of fungal organisms. Methodology: Eight sampling sites were selected for this study. The hospital main lobby was the comparison site for the three non-air-conditioned surgery wards (NACWs) while the fourth level nurse station is the comparison site for the air-conditioned wards (ACWs). Meteorologic conditions such as environmental temperature and relative humidity were also determined. Andersen air sampler was utilized to conduct the environmental indoor air sampling. A total of 98 malt extract agar supplemented with chloramphenicol (0.01%) plates were utilized for the duplicate sampling in eight sites. After three to five days of incubation at 37° C, the isolated fungal organisms were culturally and morphologically characterized. Results: Seven fungal organisms were isolated from the indoor air sampling conducted namely: Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Curvularia sp., Penicillium sp., Alternaria sp. and Rhizopus sp.). The most dominant fungal species among the NACWs was A. niger. On the other hand, A. fumigatus was the most observed isolate among the ACWs. The air-conditioned wards showed a higher number of fungal isolates. In particular, A. fumigatus and A. flavus colonies in the ACWs were evidently higher than in the NACWs. Conclusion The ubiquitous nature of the Aspergillus species and slow settling rate due to small spore size make it the most dominant fungal organism retrieved in the air sampling conducted. No strict numerical guidelines were available for the spore counts of Aspergillus species to assess contamination rate. However, according to the Health Protection Surveillance Centre, 2018, the values of CFU/m³ of most of the isolates not only by Aspergillus species showed non-compliance with the threshold level documented.
Temperature

Background and Objectives: The increase in the number of invasive Aspergillus infections has been observed among immunocompromised and hospitalized patients. In the Philippines to date, no published data focused on the prevalence of Aspergillus species or any other thermotolerant fungal species in a hospital environment. This research served as a primary study to characterize the antifungal susceptibility of environmental strains of Aspergillus fumigatus from a hospital facility against three antifungal agents and to determine the virulence of these isolates on BALB/c mice using an animal survival assay.
Methodology: Ten environmental strains of A. fumigatus were isolated from three air-conditioned wards in a medical facility using Andersen Air Sampler. The antifungal susceptibility profile of the isolates was determined against Voriconazole, Amphotericin B and Caspofungin. The virulence of these isolates was also tested on BALB/c mice using an animal survival assay. Moreover, the lung tissues of infected BALB/c mice were subjected to histopathological analyses using Gomori Methenamine Silver stain (GMS) and Hematoxylin & Eosin (H&E) stains.
Results: Etest result for antifungal susceptibility testing showed that two of the ten isolates were resistant to Amphotericin B (AF2-A and AF-3A); one isolate resistant to Voriconazole (AF2-A) and an isolate that manifested non- susceptibility to Caspofungin m(AF2-A). Epidemiological cut-off values were determined for each antifungal following the M38-A2 CLSI guidelines. BALB/c mice median survival analysis revealed that the isolate with the highest Minimum Inhibitory Concentration (MIC= 4.89 ?g/ml) for Voriconazole resulted in the most number of mortality with the least number of observation days. GMS AND H&E histopathology slides showed fungal elements embedded on left lung lobe of mice.
Conclusion: This study showed that there were strains of Aspergillus fumigatus from a hospital indoor air which were considered as resistant strains to Voriconazole, Amphotericin B, and Caspofungin (AF2-A and AF3-A). Lung tissues of infected mice showed characteristics of bronchopneumonia.

Survival Analysis ; Disk Diffusion Antimicrobial Tests