|Year : 2020 | Volume
| Issue : 3 | Page : 345-351
Direct detection of Acinetobacter baumannii by loop-mediated isothermal amplification in patients with respiratory tract infection
Seham K Khirala1, Azza A Elthoqapy1, Ragaa A Awad1, Gamal A Badr2
1 Department of Microbiology and Immunology, Faculty of Medicine for GirlsMedicine, Al-Azhar University, Cairo, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Al-Azhar University, Cairo, Egypt
|Date of Submission||18-Feb-2020|
|Date of Decision||19-Feb-2020|
|Date of Acceptance||02-Mar-2020|
|Date of Web Publication||2-Oct-2020|
MBBCh, MSC Seham K Khirala
Department of Microbiology and Immunology, Faculty of Medicine for Girls, Al-Azhar University, Cairo
Source of Support: None, Conflict of Interest: None
Background Rapid detection and treatment of Acinetobacter baumannii which is a health-care-associated pathogen that causes outbreaks and frequently encountered in ICU patients on mechanical ventilation is very important.
Aim The present study aimed to detect the frequency of A. baumannii in sputum sample by loop-mediated isothermal amplification (LAMP) assay in comparison with the different culture methods.
Patients and methods In all, 200 sputum samples and 100 tracheal aspirates (TA) were included to detect the frequency of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) pathogens by cultural methods and to detect A. baumannii from sputum sample by LAMP assay comparing its results with CHROMagar Acinetobacter and conventional culture methods referring to the rate of multidrug-resistant A. baumannii.
Results By conventional culture, positive culture was reported in 228/300 (76%) of all samples. A. baumannii and Klebsiella spp. were the most identified pathogens as they were detected in 27/145 (18.6%) and 19/145 (13%) of sputum samples and 19/83 (22%) and 43/83 (51.8%) of TA. Regarding culture on CHROMagar 46 isolates were identified as A. baumannii, 27 were from sputum sample, and 19 from TA. Out of 46 A. baumannii isolates multidrug-resistant A. baumannii were detected in 9/27 (33.3%) and 15/19 (78.9%) in sputum samples and TA, respectively. DNA of A. baumannii was detected in 28/200 (14%) by LAMP assay from sputum samples. Sensitivity and specificity of LAMP assay were 100 and 99.5% when compared with the conventional culture.
Conclusion CHROMagar Acinetobacter and LAMP assay are cost-efficient methods in comparison to conventional culture. LAMP assay is distinguished from the others for its simplicity and rapid detection of pathogens.
Keywords: Acinetobacter baumannii, antimicrobial susceptibility, CHROMagar, loop-mediated isothermal amplification assay
|How to cite this article:|
Khirala SK, Elthoqapy AA, Awad RA, Badr GA. Direct detection of Acinetobacter baumannii by loop-mediated isothermal amplification in patients with respiratory tract infection. Sci J Al-Azhar Med Fac Girls 2020;4:345-51
|How to cite this URL:|
Khirala SK, Elthoqapy AA, Awad RA, Badr GA. Direct detection of Acinetobacter baumannii by loop-mediated isothermal amplification in patients with respiratory tract infection. Sci J Al-Azhar Med Fac Girls [serial online] 2020 [cited 2020 Oct 26];4:345-51. Available from: http://www.sjamf.eg.net/text.asp?2020/4/3/345/296932
| Introduction|| |
In recent years, the Infectious Diseases Society of America has highlighted a fraction of antibiotic-resistant bacteria acronymically dubbed ‘the Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) pathogens.’ These worrisome pathogens are capable of ‘escaping’ the biocidal action of antibiotics and mutually representing new paradigms in pathogenesis, transmission, and resistance. These ESKAPE pathogens are responsible for a substantial percentage of nosocomial infections . Acinetobacter baumannii is an opportunistic pathogen affecting immunocompromised patients especially those in ICU . Its importance is due to the continuous increasing rate of drug resistance . Conventional methods used for isolation of Acinetobacter are time consuming . CHROMagar Acinetobacter is a selective medium for rapid detection of Acinetobacter and multidrug-resistant (MDR) A. baumannii . Rapid detection of A. baumannii directly from samples may be lifesaving, which can be achieved by different amplification techniques. However, loop-mediated isothermal amplification (LAMP) assay is preferred than other molecular techniques as it can be applied on crude DNA as it is not affected by the inhibitory effect of biological substances in the sample . The reaction occurs under isothermal condition as Bst or Bsm DNA polymerases with strand displacement activity are used. The LAMP has high specificity as four sets of primers identifying six distinct regions on the target gene are used. Two loop primers may be added to make the reaction more rapid and more sensitive . Besides the product of amplification gives a ladder-like pattern on gel electrophoresis and  in addition to either direct visual detection of LAMP product by turbidity due to accumulation of magnesium-pyrophosphate or by the addition of fluorescent dye such as SYBR Green .
| Aim|| |
The present study aimed to detect the frequency of A. baumannii in sputum sample by LAMP assay in comparison with the different culture methods.
| Patients and methods|| |
The study was approved by the Ethics Committee of Faculty of Medicine for Girls, Al-Azhar University and written informed consent was obtained from all participants.
A total of 300 samples [200 sputum samples from patients diagnosed with lower respiratory tract infection were collected from El Hussein University Hospital, and 100 tracheal aspirates (TA) from ICU patients were collected from Nasser Institute Hospital] in the period from October 2017 to November 2018 were included in the study.
The following reference strains were used for quality control in all work steps: A. baumannii ATCC 19606, Enterococcus faecalis ATCC 29212, and Candida tropicalis ATCC 1369. Sputum validity was assessed by means of the Geckler et al.  criteria.
Methods of the study
Aiming to detect the rate of ESKAPE pathogens, all samples were inoculated on plates of MacConkey’s and blood agar. For selective isolation of A. baumannii; samples were inoculated on CHROMagar Acinetobacter (CHROMagar, Paris, France). Identification of the isolated colonies were based on their morphologic, microscopic appearance and biochemical tests.
Aiming to select carbapenem-resistant Acinetobacter, the isolated A. baumannii were cultured on CHROMagar Acinetobacter supplemented with MDR-selective supplement (CR102).
Molecular detection of Acinetobacter baumannii
- DNA was extracted from reference strain (A. baumannii ATCC 19606) .
- Sputum samples were decontaminated by the standard protocol using N-acetyl-L-cysteine-2% NaOH and DNA was extracted by the heat shock method .
DNA amplification by loop-mediated isothermal amplification assay
Reagents used in loop-mediated isothermal amplification assay
Primer sequence  was used for the amplification of A. baumannii DNA by LAMP assay obtained from Invitrogen (Germany). The primer sequences are shown in [Table 1].
|Table 1 Primer sequence used for the amplification of Acinetobacter baumannii DNA by loop-mediated isothermal amplification assay|
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LAMP reaction was carried out in a total volume of 25 µl containing the following:
Bsm Pol buffer 1×(20 mmol/l) tris-HCl (pH 8.8 at 25 °C), 10 mmol/l KCl, 10 mmol/l (NH4)2SO4, 5 mmol/l MgSO4, 0.1% Tween 20 (Thermo Scientific-Fermentas, Vilnus, Lithuania), Betaine 0.8 M (Sigma-Aldrich, St Louis, Missouri, USA), dNTPs 1.2 mmol/l of each (Thermo Scientific-Fermentas), inner primer FIP and BIP 40 pmol of each, outer primer F3 and B3 5 pmol of each. Loop primers LF and LB 20 pmol of each, Bsm DNA polymerase 8U (Thermo Scientific-Fermentas), MgSO4 4 mmol/l and DNA template 1 µl (concentration of DNA 5 ng/µl). Tubes were incubated in a thermal cycler (63°C) for 60 min. The mixtures were then heated to 80°C for 10 min to terminate the reaction  DNA of A. baumannii ATCC 19606 were used as a positive control while deionized water was utilized instead of DNA template as a negative control.
Detection of LAMP products were done by:
- SYBR Green (1 µl of 1 : 10 stock dilution of ×10 000 concentrated in DMSO SYBR Green (Invitrogen) was added to 15 μl of LAMP products and were observed under ultraviolet illumination to observe green fluorescence .
- Gel electrophoresis 10 μl of the LAMP products were electrophoresed on a 1.5% agarose gel .
- Gene ruler 100 bp DNA ladder (0.1 μg/μl) with 1 ml 6× DNA loading buffer, with 10 chromatography-purified individual DNA bands ranging from 1000 to 100 bp molecular size.
- Data collected were reviewed; coding and statistical analysis of the collected data were done by using the Statistical Package for Social Sciences program (SPSS Inc., Chicago, Illinois, USA), version 23, for Microsoft Windows.
- Comparing groups was done using χ2: for comparison of qualitative data the level of significance was taken at P value of less than 0.05 with confidence level 95%. Sensitivity, specificity, positive and negative predicitive values, and accuracy of the test were calculated.
| Results|| |
- The mean ages of patients from whom sputum sample and endotracheal aspirate (EA) were obtained were (55.9±13.7 and 41.8±22.9 years), respectively, and chronic obstructive pulmonary disease was the most common risk factor among lower respiratory tract infection (LRTI)-infected patients 180/200 (90%).
Bacterial profile of culture on MacConkey’s agar and blood agar
Positive culture was reported in 228 samples while 72 samples were culture negative:
- Acinetobacter, Klebsiella, and Pseudomonas species were the most common isolated organisms; their rate was 20, 27, and 12.7%, respectively ([Table 2]).
Results of culture on CHROMagar
- Based on the morphological characterization of the obtained colonies, microscopic appearance, and biochemical tests ([Figure 1] and [Figure 2]), 46 (15.3%) isolates were identified as A. baumannii, 27 (13%) isolates were from sputum samples, and 19 (19%) isolates were from EA.
|Figure 1 Biochemical reactions of Acinetobacter baumannii and Acinetobacter lwoffii.|
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|Figure 2 Metallic reddish colonies of Acinetobacter baumannii on CHROMagar Acinetobacter.|
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- Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of CHROMagar were 100% when compared with MacConkey agar and blood agar ([Table 3]).
|Table 3 Validity of CHROMagar for the detection of Acinetobacter baumannii from sputum samples|
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Results of culture of Acinetobacter baumannii isolates on CHROMagar supplemented with multidrug-resistant selective supplement
- MDR A. baumannii were detected in 9/27 (33.3%) and 15/19 (78.9%) of A. baumannii isolates in sputum samples and EA, respectively. There were significant differences between frequency of detection A. baumannii in sputum and EA samples. P value of 0.002. So MDR
A. baumannii were more in ICU patients ([Table 4]).
|Table 4 Distribution of nonmultidrug-resistant Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii isolates|
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Results of loop-mediated isothermal amplification assay
Out of 200 sputum samples DNA of A. baumannii was detected in 28 (14%) of the samples by SYBR Green. The positive reaction showed color change to green, which was distinct from tubes with negative reaction that had an orange color ([Figure 3]) and by gel electrophoresis the positive reaction showed a ladder-like pattern while no bands were detected in negative reaction ([Figure 4]).
|Figure 3 LAMP product stained by fluorescent dye. LAMP, loop-mediated isothermal amplification.|
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|Figure 4 Gel electrophoresis of LAMP product. LAMP, loop-mediated isothermal amplification.|
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Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of LAMP assay was 100, 99.4, 96.4, 100, and 99.5%, respectively ([Table 5]).
|Table 5 Validity of loop-mediated isothermal amplification assay for the detection of DNA of Acinetobacter baumannii|
Click here to view
| Discussion|| |
The ESKAPE pathogens are responsible for the majority of nosocomial infections and capable of ‘escaping’ the biocidal action of antimicrobial agents .
A. baumannii is an important opportunistic nosocomial pathogen mainly affecting patients in ICUs with increasing rate of MDR isolates ,. Conventional culture requires more than 3 days for the isolation and identification of A. baumannii. CHROMagar Acinetobacter is a newly developed selective medium for the rapid isolation and identification of Acinetobacter, these media contain components that inhibit the growth of most Gram-positive and Gram-negative bacteria. Colonies obtained are identified within 18–24 h of inoculation relaying on color-based appearance .
In addition, CHROMagar Acinetobacter supplemented with MDR-selective supplement (CR102) can detect carbapenem-resistant Acinetobacter directly without the need of doing antibiotic susceptibility testing and can be used instead of PCR used for resistant gene detection.
In this study, CHROMagar Acinetobacter was validated in comparison with MacConkey agar for the detection of A. baumannii. The sensitivity and specificity were 100%.
The performance of CHROMagar Acinetobacter supplemented with MDR-selective supplement which detected 24 (8%) MDR A. baumannii isolates from samples was evaluated in comparison with antibiotic susceptibility testing which detected 23 (7.6%) MDR A. baumannii isolates so there was no significant difference between them.
Also Ghaima et al.  and Soltani et al.  reported that the sensitivity and specificity of CHROMagar Acinetobacter for the isolation of A. baumannii was 100%.
This is in accordance to Ajao et al. , who found that CHROMagar Acinetobacter was 100% sensitive for Acinetobacter isolates compared with MacConkey agar.
Another study evaluated the use of MDR CHROMagar Acinetobacter for the detection of MDR Acinetobacter species in nasal and rectal surveillance cultures. There was a 100% concordance between CHROMagar Acinetobacter and VITEK 2 system in terms of identification; they had the same results in antimicrobial susceptibility using E-test strips .
Although Ghaith et al.  uses PCR as a reference method for the identification of blaOXA-51 gene; they also found that the sensitivity and specificity of MDR CHROMagar Acinetobacter were 96.4 and 100%.
Gordon and Wareham  found that the sensitivity and specificity of MDR CHROMagar Acinetobacter was 91.7 and 89.6% when compared with the PCR.
PCR or real-time PCR methods require high costs and expensive equipment, also using the Taq polymerase require pure DNA for the preparation of reaction mixtures. LAMP assay is a simple, rapid, specific, and cost-effective method that can detect the pathogen directly from the sample (not needing extensive DNA purification) within 2 h, tolerance to changes in conditions such as elongation period and prolonged warming during reagent preparation, so LAMP is very beneficial especially in limited resource countries .
In our study, LAMP assay detect DNA of Acinetobacter baumannii in 14% of sputum samples.
A study by Zhang et al.  has shown that DNA of A. baumannii was detected in 19.2% of sputum samples by LAMP assay which is higher than our result; a difference that may be attributed to the fact that the prevalence of A. baumannii varies with different countries.
In our study the sensitivity and specificity of LAMP assay were 100 and 99.4% when compared with the conventional culture method.
The same results were obtained by Hou et al. , who demonstrated the higher detection rate by the LAMP assay than the bacterial culture demonstrating that the LAMP assay was 100% specific and 95.50% sensitive when compared with DNA-sequencing analysis.
Also Zhang  found that LAMP assay showed significantly high detection rate when compared with blood culture for the detection of pathogens from clinical specimens of sepsis and nonsepsis patients.
Yamazaki et al.  found that sensitivity and specificity of LAMP assay for the isolation of Campylobacter jejuni and Campylobacter coli directly from stool samples was 81.3 and 96.6% compared with the conventional culture. Sensitivity and specificity of LAMP in this study is lower than our study which may be due to the presence of inhibitors in stool samples.
Real-time LAMP showed 98.9% sensitivity and 75.0% for the detection of A. baumannii when compared with VITEK 2 system sensitivity and also lower than our study may be due to the use of nonspecific dye .
Conclusion and recommendations
CHROMagar Acinetobacter is a highly sensitive and specific method for the detection of A. baumannii. MDR CHROMagar can detect MDR Acinetobacter spp. within 17–24 h. LAMP assay is also highly sensitive and specific for the detection of A. baumannii and is more rapid than other culture methods and has many advantages over other molecular techniques.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]