Study design, site and duration
A hospital-based prospective cross-sectional study was conducted in Antiretroviral Treatment (ART) center of Sukraraj Tropical and Infectious Disease Hospital, one of the tertiary care hospitals with 100 beds capacity in Kathmandu, Nepal from February to August 2019. The hospital was the first to start ART service in Nepal in 2004. Around 4200–4500 PLHA visit the ART center for therapy annually. Sample collection from the participants and processing were done in the same hospital. In addition, further processing of plasmid DNA extraction and gene detection were done in the Central Department of Microbiology, Tribhuvan University, Kathmandu.
Inclusion and exclusion criteria
All age groups of both sexes living with HIV, under ART and with LRTIs confirmed by a physician after physical examination, blood investigations including CBC and chest x-ray, were enrolled n this study. Sputum samples and the demographic information from the participants were collected only after obtaining written consent. However, the patients infected with tuberculosis were excluded from the study.
The calculated sample size was 255 based on the 21% prevalence rate of bacterial LRTIs in PLHA as reported by Kandati et al. . Therefore, for this study, 263 sputum samples were collected.
Sample collection, transportation and processing
Sputum sample were collected from the participants using sterile vials with a wide mouth and tight lid. Participants were instructed first to take a deep breath and then to expectorate a cough . The collected sputum samples were transported to the Microbiology laboratory for further processing. The quality of sputum was checked macroscopically for the presence of mucopurulent part before accepting the sample.
Mucopurulent sputum was cultured on MacConkey Agar (MA), Blood Agar (BA), and Chocolate Agar (along with 10-unit bacitracin disk) and incubated at 37 °C for 24 h. Incubated plates were examined for the presence of distinct colonies, and identification was made using standard microbiological protocols, including their colony morphology on different culture media, microscopically by Gram staining and various biochemical tests .
For identification of H. influenzae, a satellitism test was performed. A loopful of suspected colonies of H. influenzae was mixed in about 2 ml of sterile physiological saline. Using a sterile swab stick, the bacterial suspension was inoculated on a plate of blood agar, and a pure culture of S. aureus was streaked across the inoculated blood agar plate. It was then incubated in a CO2 enriched atmosphere at 35–37 °C for 18–24 h. The culture plate was examined for growth and satellite colonies .
Antibiotics susceptibility test
Out of 67 bacterial isolates from sputum culture, only 53 isolates excluding H. influenzae (n = 14) were processed further. Antibiotic susceptibility patterns of those organisms were performed using the modified Kirby Bauer disk diffusion method recommended by CLSI 2019 . The antibiotics used were amoxicillin (10 µg), amoxicillin/clavulanic acid (AMC, 20/10 mcg), cefepime (CPM, 30mcg), ceftazidime (CTZ, 30 mcg), ceftriaxone (CTR, 30 mcg), chloramphenicol (C, 30 mcg), ciprofloxacin (CIP, 5 mcg), colistin (CL 10 mcg), co-trimoxazole (COT, 25 mcg), gentamycin (GEN, 10 mcg), imipenem (IMP, 10 mcg), piperacillin/tazobactam (PIT, 100/10 mcg), polymyxin-B (PB, 300 units) and tetracycline (TET, 30 mcg). The isolates resistant to at least one agent in ≥ 3 antimicrobial categories were considered MDR . Subsequently, the prevalence of MDR bacteria was determined.
Screening and confirmation of extended-spectrum β-lactamase (ESBL)
The isolates resistant to antibiotics—ceftazidime (30 µg) or ceftriaxone (30 µg) were considered as potential ESBL producers. The confirmatory test was performed by double disk diffusion method using ceftazidime and ceftazidime clavulanic acid. More than 5 mm zone of diameter around ceftazidime-clavulanic acid disc than ceftazidime disc alone was confirmed an ESBL producer .
Detection of metallo β-lactamase (MBL)
Two imipenem discs were placed on an MHA plate inoculated with a test organism (bacterial density equivalent to 0.5 McFarland Standard). A 5 µl of EDTA (0.5 M, pH = 8.0) solution was added to one of the imipenem discs and incubated for 16–18 h at 37 °C. An increased zone of diameter (> 7 mm) around the imipenem and EDTA disc than the imipenem alone was confirmed MBL positive .
Bacterial plasmid DNA extraction
Plasmid DNA from MDR isolates was extracted by the alkaline lysis method followed by purification with phenol–chloroform. The isolated plasmid DNAs were visualized on 0.8% agarose gel electrophoresis with 0.2 μg/ml concentration of ethidium bromide as described by Sambrook, 1989 and Thapa Shrestha and Adhikari 2014 [12, 13].
Molecular detection of bla
CTX-M and bla
A set of primers for each gene was selected from the previous studies and verified on NCBI BLAST. A set of primers (Forward: 5′-TTTGCGATGTGCAGTACCAGTAA-3′ and reverse: 5′-CTCCGCCTGCCGGTTTTAT-3′) as described in Edelsteint et al.  were used for amplification of blaCTX-M gene. Similarly, a set of primers (Forward: 5′-GAGACAATAAGGGTGGTAAAT-3′ and reverse: 5′-AGAAGTAAGTTGGCAGCAGTG-3′) as mentioned in Sharma et al. 2013 were used to amplify blaTEM gene . A conventional PCR was used to amplify the blaTEM and blaCTX-M genes. A PCR reaction mixture was prepared containing 12.5 µl master mix (Qiagen), 0.5 µl of each primer, 3 µl template DNA and 8.5 µl PCR grade water . PCR amplification was run at initial denaturation of 95 °C for 15 min; followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 55 °C for blaTEM genes and 56 °C for the blaCTX-M gene for 30 s, extension at 72 °C for 3 min and a final extension at 72 °C for 10 min. The PCR products were analyzed on 1.5% agarose gel electrophoresis with 0.2 μg/ml concentration of ethidium bromide and visualized under UV transilluminator.
Control strain of E. coli (ATCC 25922) and P. aeruginosa (ATCC 27853) were used as positive controls for ESBL and MBL producing strains, respectively. For PCR, a previously harvested plasmid DNA containing target genes were used as positive control and nuclease-free water as negative control.
Data management and analysis
All data collected were analyzed using SPSS-21 software. The Chi-square test was used to assess the association of different variables. p-value < 0.05 was considered as significant.