Antibiotic Susceptibility Pattern of Clinical Isolates of Methicillin Resistant Staphylococcus Aureus in Peshawar, Pakistan
Ayesha Asif1, Muhammad Asghar2, Habib Ullah Khan3, Ihteshamul Haq*4, Syed Luqman Shuaib*5, Fawad Khalid6, Sidra Khan1, Shah Zaman7, Muhsina Haq7, Awais Khan4, Noor
Rehman8
Department of Microbiology Shaheed Benazir Bhutto Women University Larama Peshawar kp,Pakistan
Department of Microbiology Khyber Medical College (KMC) and Khyber Teaching hospital (KTH) Peshawar Kp Pakistan.
Biotechnologist in Khyber Medical College (KMC) and Khyber Teaching hospital (KTH) Peshawar Kp Pakistan.
Department of Biotechnology and Genetic Engineering, Hazara University, Mansehra KP Pakistan.
Department of Microbiology Khyber Medical College (KMC) Peshawar Kp Pakistan Saidu Medical College Saidu Sharif Swat KP Pakistan.
Department of Pharmacology Peshawar Medical College Peshawar KP Pakistan.
Department of Microbiology Khyber Teaching Hospital(KTH) Peshawar Kp Pakistan Corresponding Author: Syed Luqman Shuaib*5
Email: [email protected] Co-Corresponding Author: Ihteshamul Haq*4
Email: [email protected]
Abstract:
The Staphylococcus aureus are facultative anaerobic gram-positive cocci that arrange in grape like bunch of irregular clusters. On the base of enzyme coagulase, the organismhaspotential to clot blood plasma; staphylococci are separated into coagulase positive staphylococci and coagulase negative staphylococci. Coagulase-positive staphylococci include pathogenic organism named S. aureus. MRSA is recognized as a major cause of nosocomial infections ranging from minor skin infection to life threatening disease. The emergence of MRSA strains is
an alarming situation for both hospitalized and non-hospitalized patients.The current study was aimedto find the prevalence and antibiotic susceptibility pattern of MSSA and MRSA from clinical isolates. A total of 100 clinical isolates of Staphylococcus aureus was collected from indoor patients of Khyber Teaching Hospital Peshawar. The isolation and identification of these clinical isolates were done using standard microbiological methods as per CLSI guidelines. The Antibiotic susceptibility pattern was done by using disk diffusion method. Out of 100 clinical isolates, 18(18%) were MRSA and 82(82%) were MSSA. Prevalence of MRSA was same in male and female. MSSA showed higher susceptibility to doxycycline (98.0%) while AMC (29.0%) showed lowest susceptibility against S. aureus. MRSA was higher sensitive to Vancomycin and Amikacin (94.4%) but resistant to cefoxitin (100%). Thecurrent study concludes that 18% isolates were MRSA in indoor patients of MTI KTH while the remaining isolates were MSSA.
Keywords: MRSA, Bacteria, MSSA, Antibiotics, Peshawar, coagulase, Staphylococcus aureus Introduction:
Methicillin-resistant Staphylococcus aureus (MRSA) is the extremely frequent cause of nosocomial infections around the world. The World Health Organization (WHO) must be classified MRSA in accordance with a global high priority pathogen list to help promote research and development the pipeline for the finding of novel antimicrobials [1]. Staphylococcus aureus are facultative anaerobic gram-positive cocci, usually arranged in grape like bunch of irregular clusters. The S. aureus are classified according to activity of enzyme coagulase the organism has ability to clot blood plasma, staphylococci are separated into coagulase positive staphylococci and coagulase negative staphylococci [2]. Coagulase-positive staphylococci include pathogenic organism named S. aureus. In the early 1960s, beta-lactamase resistant semi-synthetic penicillin became available for clinical use; it was effective for a while but ended with the emergence of Methicillin (oxacillin) Resistant Staphylococcus aureus (MRSA) [3]. Worldwide, Methicillin- resistant Staphylococcus aureus (MRSA) is a major a etiology of nosocomial infections. The emergence of MRSA is an alarming situation in the community for both hospitalized and non- hospitalized patients. Earlier studies have found that such infections have distinctive strain,
great concern for the clinicians, as the mortality and morbidity of disease is quite high [6]. The irrational use of antibiotic is one of the major causes of MRSA infection and prolonged hospital stay. [7]. Reservoirs are the Infected and colonized patients and primarily it transmits through contaminated hands [8]. there number of MRSA strains are resistant to antimicrobial drugs, but the glycopeptide antibiotic is still working against the resistant strains of S. aureus including vancomycin [9] Staphylococcus aureus and MRSA are normally found on the skin and mucous membranes of healthy individuals; thus, humans are the major reservoir for these organisms [10,11]. According to research, it is estimated that approximately half of all adults are colonized, and up to 15% of the population persistently carry S. aureus in the anterior nares. Some individuals are more vulnerable to higher rates of S. aureus colonization (up to 80%), such as health care workers, persons who use needles on a regular basis (i.e., diabetics and intravenous (IV) drug users), hospitalized patients, and immune compromised individuals. Staphylococcus aureus can be transferred from one person to another either by direct contact, by infected droplets from sneezing or coughing, or by fomites [12,13,14]. Staphylococcus aureus found in the normal flora of intact skin. S. aureuspersist as a hospital acquired and community-acquired pathogen and causes public health concern. Mucous membranes have recently got attention as a potential pathogen, specifically for nosocomial infections [15,16]. S. aureus is important pathogenic bacterium responsible for minor skin infections such as folliculitis, furuncles, andabscesses to life-threatening diseases such as meningitis, pneumonia, endocarditis, toxic shock syndrome (TSS), and septicemia [17,18]. The aim of current to find the prevalence of MRSA isolated from clinical patients Isolation and identification of MRSA from clinical isolates of infected patients and observe the antibiogram of S. aureus against selected antibiotics in KTH hospital Peshawar.
Methodology:
Study Site:
This study was conducted in department of pathology at KTH Peshawar. from December 2019 to March 2020 in microbiology section.
Sample collection:
A total 650 clinical samples were collected from indoor patients. Different types of specimens were collected including pus, fluids, sputum, and blood collected in sterilized bottles and swabs are processed in microbiology laboratory in Khyber teaching hospital (KTH). These collected samples were labeled properly.
Isolation and identification of clinical specimens:
All the samples of S. aureus were cultured on MacConkey and sheep blood agar and incubated for 24hrs at 37C for the bacterial growth. These bacterial isolates were then subjected by performing standard microbiological techniques such as Gram staining and biochemical tests.
The isolates were further processed to culture sensitivity by disc diffusion method using different selected antibiotics on nutrient agar.
Media preparation
Media sterilization was done by using autoclave at temperature 121cand maintained for 15 to 20 mints. The media used for culturing are blood agar and MacConkey agar, Nutrient agar, Nutrient broth media. MacConkey agar is differential and selective medium used for the isolation and differentiation of gram-negative rods. It differentiates lactose and non-lactose fermenting bacteria the procedure of composition as shown in Table.No.1.
Table.No.1: Composition of MacConkey Agar:
Peptone 20.0
Bile salt 31.5
Sodium chloride 5.0
Neutral red 0.03
Crystal violet 0.001
Agar 15.0
PH 7.1+-0.2at 25C
Gram staining:
Gram staining had been done for microscopic analysis which is differential method for differentiation of gram negative and gram-positivebacteria. Water drop was put on slide and with the help of loop colony was streaked onto the slide in circular motion to make a thin layer. The smear was heat fixed. Crystal violet dye was applied on the heat fixed smear and remained for 60 seconds and was then washed under running tap water.Then the slide was flooded with iodine solution and remain for 60 seconds, and the slide was then washed with tap water.Decolorized with alcohol for 15 seconds and again washed off.Safranin (counter stain) was added for 30 seconds giving red\pink color to the background. Gram positive and negativebacteria as shown in Figure.No.1,
Figure.N0.1: Gram staining of positive and Negative Bacteria Biochemical tests:
For further identification and chemical analysis of positive isolates, following biochemical tests were performed: Catalasetest, Coagulase test, MSA (Mannitol salt agar).
Catalase test:
Catalase is an enzyme that break down hydrogen peroxide into water and oxygen. Thecatalase test is used to find out the catalyze enzyme presence.On a clean slide transfer a small amount of bacterial colony, dry glass slide using inoculating loop. Positive result showed bubbles production.while Negative results showed no production of bubbles.
Coagulase test:
Coagulase test is used to differentiate staphylococcus aureus (positive) from coagulase negative staphylococcus. Coagulase is an enzyme and cause plasma to clot by converting fibroin (soluble) to fibrin (insoluble). A drop of saline on to the slide.With the wire loop transfer small amount of bacterial colony on a slide in each drop to make two thick suspensions.Add drop of human plasma into the staphylococcus aureus on a slide for clumping of the organism within 10 seconds.
Mannitol salt agar (MSA):
Mannitol salt agar (MSA) is used as a selective and differential medium for isolation of staphylococcus aureus from clinical and non-clinical specimens.Mannitol salt agar 111 g was suspended in 1 liter of distilled water and boil it until it dissolves completely. ThenSterilize by autoclaving at 121C for 15 minutes as shown in Table.No.2.
Table.No.2: Composition of Mannitol salt agar (MSA)
Lab-lemon powder 1.0
Peptone 10.0
Mannitol 10.0
Sodium chloride 75.0
Phenol red 0.025
Agar 15.0
PH 7.5+-0.2 at 25C
Antimicrobial susceptibility testing:
Antibiotic susceptibility pattern was determined by commonly used antibiotic by disc diffusion method different antibiotic are used Take colonies from an agar plate with a wire loop and transferred to the tube containing 4 to 5 ml of broth medium.The broth culture was then incubated at 37OC for 2 to 8 hr.Take a dry nutrient agar plate and poured the broth containing colonies and was incubated for 15 mints at 37c.Placed sensitivity disk on the surface of nutrient agar at 24mm apart. After that plate was placed at 37OC in an incubator.After 24 hours incubation the plates was examined.
Results:
A total of 650 samples were collected from infected patients visiting to MTI, KTH, Peshawarincluding samples urine, pus, wound, sputum, fluids. While total of 100 samples were collected of MRSA strains were obtained from pus (38.9%), followed by wounds (16.7%) sputum (5.6%), Fluids (11.1%), others (27.8%). as shown in Table.No.3 and Figure.N0.2.
Table.No.3: Distribution of different types of samples collected from infected patients Different Samples Male cases Female cases Total samples Percentage
Pus 12 13 25 25.0
Wounds 5 5 10 10.0
Sputum 3 1 4 4.0
Fluids 8 6 14 14.0
Others 25 22 47 47.0
Total 53 47 100 100
Figure.No.2: Distribution of different types of samples Prevalence and Gender wise distribution of clinical isolates:
A total of 650 samples were collected among which 348 were collect from male patients and 302 were collect from female. Of the samples, 100(15.3%) isolates yielded the growth of S. aureus.
Among positive isolates, 53 isolates were male and 47 were found in female patients. While in MSRA total 100 samples were collected 53 were male and 47 were females. Out 100 patients 18 are positive isolates the growth on MRSA. Among MRSA positive isolates, 9 isolates were male and 9 were found in female patients as shown in Table.N0.4 and Figure.No.3.
Table.No.4: Gender wise distribution of S. aureus positive isolates among infected patients (n=100).
Gender Total case Total negative case Total positive case
Male 348 295 53
Female 302 255 47
Total 650 550 100
3 2 1 0 3 9
4 7 1 3 0 1 2 2 2 5 9
18 38.9
16.7
5.6 11.1
27.8
100
-20 0 20 40 60 80 100 120 140
Pus Wounds Sputum Fluids Others Total
Male cases Female cases Total no of samples Percentage
Figure.No.3: Gender wise distribution of MRSA from clinical isolates Different age group:
S. aureus isolated from infected patients was distributed into different age groups. The highest prevalence of S. aureus was observed in the age group of 0-10 years (8.2%) followed by the age groups 11-20 years (4.4%), 21-40 years (7.1%), 41-60 years (4.6%) while the lowest prevalence of S. aureus was recorded in the age group of 61 and above years (3.0%). While MRSA isolated from infected patients was distributed into different age groups. The highest prevalence of MRSA was observed in the age group of 41-60 years (33.3%) followed by the age groups 0-10 years (27.8%), 11-20, 61 and above years (16.7%), while the lowest prevalence of MRSA was recorded in the age group of 21-40 years (5.6%) as shown in Table.No.5 andFigure.No.4.
Table.No.5: Different age group of patients among MRSA isolates
Different age group Male cases Female cases Frequency Percentage
0 – 10 3 2 5 27.8
11 – 20 3 0 3 16.7
21- 40 0 1 1 5.6
53
44
9 47
38
9 100
82
18
0 20 40 60 80 100 120
Total case Total positive case of S.aureus Total positive case of MRSA
Male Female Total
41 – 60 2 4 6 33.3
61 and above 1 2 3 16.7
Total 9 9 18 100
Figure.No.4: Different age group of patients among S. aureus isolates Antibiogram of staphylococcus aureus in clinical isolates:
The present Research can screen 100 clinical samples isolates of staphylococcus aureus from different clinical samples. All the S. aureus isolates were tested against the selected antibiotics by using the standard protocol of CLSI guidelines. The antibiotic doxycycline showed highest sensitivity (98.0%) against the S. aureus. susceptibility to other antibiotics which were tested was as follows AK (97.0%), CTX (95.0%), LZD (93.0%), TEC (92.0%), V (92.0%), TGC (91.0%), CD (89.0%), FC (89.0%), CFC (85.0%), E(83.0%), FOX(82.0%), CRD(77.0%), CIP(68.0%), AMP(32.0%). AMC (29.0%) showed lowest susceptibility. While the highest resistivity pattern was observed in AMC (71.0%) followed by AMP (68.0%), CIP (32.0%), AK (3.0%), CRD (23.0%), DO (2.0%), FOX (18.0%), E (17.0%), CFC (15.0%), FC (11.0%), CD (11.0%), TGC (9.0%), TEC(8.0%), V(8.0%), CTX(5.0%). LZD (7.0%) showed the lowest resistivity against S.
17 11 10 9 6
53
13 5
16 8 5
47 30
16
26
17 11
100
30
16
26
17 11
100
0 20 40 60 80 100 120
0 – 10 11 – 20 21- 40 41 – 60 61 and Above Total
Different age group Male cases Female cases
Frequency
Frequency Percentage
different clinical samples. All the MRSA isolates were tested against the selected antibiotics by using the standard protocol of CLSI guidelines. The antibiotic Vancomycin and Amikacin showed highest sensitivity (94.4%) against the MRSA. Sensitivity to other antibiotics which were tested was as follows TEC, DO and FC (88.9%), CFC (83.3%), CIP, CTX, TGC (72.2%), LZD, CRD,E,CD (66.7%), AMP(50%), AMC(38.9%), and FOX (0%) showed lowest susceptibility. While the highest resistivity pattern was observed in FOX (100%) followed by AMC (61.1%), AMP (50%), LZD, CRD, CD and E (33.3%), CTX, CIP, TGC (27.8%), CFC (16.7%), TZP, FC, DO(11.1%), AK and V(5.6%) showed the lowest resistivity against MRSA as shown in Figure.No.5.
Table.N0.6: Antibiogram of staphylococcus aureus in clinical isolates
Antibiotics Sensitive Percentage Resistant Percentage P value
AMC 29 29.0 71 71.0 246
AMP 32 32.0 68 68.0 655
TEC 92 92.0 8 8.0 575
CTX 95 95.0 5 5.0 748
FC 89 89.0 11 11.0 454
AK 97 97.0 3 3.0 630
DO 98 98.0 2 2.0 179
CIP 68 68.0 32 32.0 400
FOX 82 82.0 18 18.0 778
LZD 93 93.0 7 7.0 577
TGC 91 91.0 9 9.0 389
CRD 77 77.0 23 23.0 046
CD 89 89.0 11 11.0 241
CFC 85 85.0 15 15.0 274
E 83 83.0 17 17.0 032
V 92 92.0 8 8.0 575
Figure.No.5: Antibiogram of MRSA in clinical isolates Discussion
TheMRSA is a major issue faced by the medical sciences in the current era worldwide. The emergence of MRSA strains is an alarming situation for both hospitalized and non-hospitalized patients. Earlier studies have found that such infections have distinctive strain, virulence, and epidemiologic properties [19,20]. MRSA causes some serious infections, which is a great deal for the physicians, being associated with increase morbidity and mortality [21]. The results of the current study revealed that 18% isolates were found to be MRSA strains a total 100 isolates. Out of 18 MRSA isolates, 9 were from male and 9 from female cases. The high prevalence rate of 31% was observed in other study and in India the incidence rate of MRSA (31.8%) was reported as compared to this study [22,23]. The high prevalence of 46% was observed in other study than our findings [24]. In another study in Nagpur the rate of MRSA was 19.5% which is like this study [25]. Above studies clearly shows that prevalence of MRSA varies from one setting to
7 9
16
13 16
17 16
13
0 12
13
12 12 15
12 17
11 9
2 5
2 1 2
5 18
6 5
6 6
3 6
1
AMC AMP TZP CTX FC AK DO CIP FOX LZD TGC CRD CD CFC E V
Sensitive Resistant
it might increase with time. In the present study the prevalence rate of MRSA was higher in age group of 41-60 year (33.3%) and prevalence rate was lower in age group of 21-40 year (5.6%).
Majority of MRSA strains were obtained from pus (38.9%), followed by wounds (16.7%) sputum (5.6%), fluids (11.1%), others (27.8%). However, no inference can be drawn from these values, due to low sample size. In the current study, Cefoxitin disc was used for the identification of MRSA i.e., recommended method for the detection of MRSA strains in clinical specimens [26]. MSSA isolates were show higher sensitivity to doxycycline (98.0%) followed by AK (97.0%), CTX (95.0%), LZD (93.0%), V (92.0%), AMP (32.0%), AMC (29.0%) showed lowest susceptibility. While the highest resistivity pattern was observed in AMC (71.0%) followed by AMP (68.0%), CIP (32.0%), AK (3.0%), CRD (23.0%), CTX (5.0%), LZD (7.0%) showed the lowest resistivity against S.aureus.MRSAisolates were show higher sensitivity to Vancomycin and Amikacin (94.4%) followed by Fusidic acid, doxycycline (88.9%), Cefachlor (83.3%), Ciprofloxacin (72.2%) and AMC (38.9%), FOX(0%) showed the lowest susceptibility . On the other hand MRSAshowed significant amount of resistant against FOX (100%) followed by AMC (61.1%), AMP (50%), LZD, CRD, CD and E (33.3%), CTX, CIP, TGC (27.8%), CFC(16.7%), TEC, FC, DO(11.1%), AK and V(5.6%) showed the lowest resistivity against MRS. The same results of MRSA isolates were observed in a study in which high resistance was recorded;
Ciprofloxacin (73%), Ofloxacin (71%) and Erythromycin (67%). This is reliable with previous study completed at Ayub Medical Complex [27]. This may be due to the misuse of these antibiotics in daily practice at local hospitals. The results of our study are in accordance with a study carried out at Aga Khan University in 2009 which showed variable susceptibility pattern with high resistance rates to tetracycline (82%), clindamycin (79%), cotrimoxazole (59%), and rifampicin (50%). Resistance to chloramphenicol (10%) and Fusidic acid (9%) was low [28]. A study carried out at Lahore in 2009 showed that only 4% of MRSA isolates were sensitive to Fluoroquinoles, whereas 38% of isolates were found to be sensitive in our study [73].
Conclusion:
The current study concluded that 18% isolates were MRSA while the remaining isolates were MSSA. MRSA isolates were show higher sensitivity to Vancomycin and Amikacin (94.4%) followed by Fluidic acid, doxycycline (88.9%), Cefaclor (83.3%) while resistant against FOX (100%), AMC (61.1%), AMP (50%), CRD, CD and E (33.3%) and CTX, CIP (27.8%). Effective
antimicrobial activity as well as cost effectiveness should be considered in drugs prescribed for MRSA infections. Oral dosing options for linezolid can allow earlier discharge of hospitalized patients and minimize the chances of MRSA emergence. Good hospital infection control measures prove to be the main stay against these infections because antibiotics can never be an effective alternative to good medical practice.
References:
1. Asokan, G. V., Ramadhan, T., Ahmed, E., & Sanad, H. (2019). WHO global priority pathogens list: a bibliometric analysis of Medline-PubMed for knowledge mobilization to infection prevention and control practices in Bahrain. Oman medical journal, 34(3), 184.
2. Moroni, P., Pisoni, G., Cremonesi, P., & Castiglioni, B. 18 Staphylococcus Aureus.
3. Tyagi, A. (2007). Phenotypic and Molecular Characterization of Staphylococcus aureus Isolated from Clinical Specimens and Food Articles of Animal Origin (Doctoral dissertation, Aligarh Muslim University).
4. Aires de Sousa, M., &Lencastre de, H. (2004). Bridges from hospitals to the laboratory:
genetic portraits of methicillin-resistant Staphylococcus aureus clones. FEMS Immunology &
Medical Microbiology, 40(2), 101-111.
5. Kluytmans, J. A. N., Van Belkum, A., &Verbrugh, H. (1997). Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clinical microbiology reviews, 10(3), 505-520.
6. Tarai, B., Das, P., & Kumar, D. (2013). Recurrent challenges for clinicians: emergence of methicillin-resistant Staphylococcus aureus, vancomycin resistance, and current treatment options. Journal of laboratory physicians, 5(02), 071-078.
7. Kulkarni, A. P., Nagvekar, V. C., Veeraraghavan, B., Warrier, A. R., Ts, D., Ahdal, J., &
Jain, R. (2019). Current perspectives on treatment of Gram-positive infections in India: what is the way forward?. Interdisciplinary perspectives on infectious diseases, 2019.
8. Muto, C. A., Jernigan, J. A., Ostrowsky, B. E., Richet, H. M., Jarvis, W. R., Boyce, J. M., &
Farr, B. M. (2003). SHEA guideline for preventing nosocomial transmission of multidrug- resistant strains of Staphylococcus aureus and enterococcus. Infection Control & Hospital Epidemiology, 24(5), 362-386.
9. Perl, T. M. (1999). The threat of vancomycin resistance. The American journal of medicine, 106(5), 26-37.
10. Taylor, T. A., &Unakal, C. G. (2017). Staphylococcus aureus.
11. Kluytmans, J. A. J. W. (2010). Methicillin-resistant Staphylococcus aureus in food products:
cause for concern or case for complacency?. Clinical microbiology and infection, 16(1), 11- 15.
12. Vandecasteele, S. J., Boelaert, J. R., & De Vriese, A. S. (2009). Staphylococcus aureus infections in hemodialysis: what a nephrologist should know. Clinical journal of the american society of nephrology, 4(8), 1388-1400.
13. Anwar, F., Khan, M., Salman, M., Ahmad, S., Ullah, F., Khan, J., ... & Abbas, M. (2021).
Seroprevalence of hepatitis B virus in human population of district Buner Khyber Pakhtunkhwa Pakistan. Clinical Epidemiology and Global Health, 10, 100688.
14. Conceição, T., de Lencastre, H., & Aires-de-Sousa, M. (2017). Carriage of Staphylococcus aureus among Portuguese nursing students: a longitudinal cohort study over four years of education. PloS one, 12(11), e0188855.
15. Gajdács, M. (2019). The continuing threat of methicillin-resistant Staphylococcus aureus. Antibiotics, 8(2), 52.
16. Akindolire, M. A., Babalola, O. O., &Ateba, C. N. (2015). Detection of antibiotic resistant Staphylococcus aureus from milk: a public health implication. International journal of environmental research and public health, 12(9), 10254-10275.
17. Al-Mebairik, N. F., El-Kersh, T. A., Al-Sheikh, Y. A., & Marie, M. A. M. (2016). A review of virulence factors, pathogenesis, and antibiotic resistance in Staphylococcus aureus. Reviews in Medical Microbiology, 27(2), 50-56.
18. Rehman, A., Haq, I., Asghar, M., Afridi, G. Z., & Faisal, S. (2020). Sero-epidemiological Identification of Dengue Virus in Individuals at District Shangla, Khyber Pakhtunkhwa. Pakistan. J Biomedical Sci, 9(3), 10.
19. Al-Mebairik, N. F., El-Kersh, T. A., Al-Sheikh, Y. A., & Marie, M. A. M. (2016). A review of virulence factors, pathogenesis, and antibiotic resistance in Staphylococcus aureus. Reviews in Medical Microbiology, 27(2), 50-56.
20. Haq, I., Ullah, R., Din, M., Ahmad, S., Anwar, F., Ali, M., & Khan, H. U. (2020).
Unrecognized HIV infection in asymptomatic volunteer blood donors at district Peshawar, Khyber Pakhtunkhwa, Pakistan. New Microbes and New Infections, 35, 100685.
21. Anwar, F., Ahmad, S., Haroon, M., Haq, I. U., Khan, H. U., Khan, J., ...& Shah, I. A. (2019).
Dengue virus epidemics: A recent report of 2017 from district Mardan, Khyber Pakhtunkhwa province, Pakistan. International Journal of Mosquito Research, 6(1), 46-49.
22. Anwar, F., Tayyab, M., Salman, M., Abdullah, Din, M., Khan, J., & Haq, I. (2020). Dengue outbreak 2018 in district Shangla KPK; clinical features and laboratory markers of dengue virus infection. Future Virology, 15(10), 693-699.
23. Din, M., Anwar, F., Ali, M., Yousaf, M., & Ahmad, B. (2021). Chemiluminescent- microparticle-immunoassay-based detection and prevalence of human immunodeficiency virus infection in Islamabad, Pakistan. Archives of Virology, 166(2), 581-586.
24. Bouchiat, C., El-Zeenni, N., Chakrakodi, B., Nagaraj, S., Arakere, G., & Etienne, J. (2015).
Epidemiology of Staphylococcus aureus in Bangalore, India: emergence of the ST217 clone and high rate of resistance to erythromycin and ciprofloxacin in the community. New microbes and new infections, 7, 15-20.
25. Bashir, Z., Ahmad, S. U., Kiani, B. H., Jan, Z., Khan, N., Khan, U., ... & Mahmood, T.
(2021). Immunoinformatics approaches to explore B and T cell epitope-based vaccine designing for SARS-CoV-2 Virus. Pak. J. Pharm. Sci, 34(1), 345-352.
26. Anwar F, Tayyab M, Khan J, Haq I. COVID-19 and taking care and protection of patients with intellectual disabilities, need special care and equity. Int J Clin Virol. 2020; 4: 116-117.
27. Qamar, Z., Anwar, F., Ahmad, R., Haq, I., Khan, A. M. K., Hussain, R., ... & Khan, J.
(2021). Prevalence of Hepatitis C virus and determination of its genotypes in subjects of Tehsil Daggar District Buner, KP, Pakistan. Clinical Epidemiology and Global Health, 12, 100809.
28. Ahmad, I., Jan, H., Salman Munir Malik, Q. A., Haq, I., Hassan, I., Ullah, I., ... & Hussain, A. (2021). Comparative Evaluation of ALT & AST Levels of Hepatitis B and C Infected Pregnant Women in Lahore, Pakistan. Annals of the Romanian Society for Cell Biology, 25(6), 19829-19837.
29. Kaleem, F., Usman, J., Hassan, A., Omair, M., Khalid, A., & Uddin, R. (2010). Sensitivity pattern of methicillin resistant Staphylococcus aureus isolated from patients admitted in a tertiary care hospital of Pakistan. Iranian journal of microbiology, 2(3), 143.
