The current study included 58 cases of Neutropenic fever in 58 adult leukemia patients who underwent chemotherapy and were enrolled in the Basrah Center for Oncology and Hematology from October 2021- June 2022.

This study conducted according to the approvals of the Iraqi Ministry of Health / Basra Health Department No 58/2021 as of date 24/10/2021, task facilitation to Al-Sader Teaching Hospital and Basrah Center for Oncology and Hematology No 749 as of date 24/10/2021.

The patients were informed about the purpose of blood sampling that was to be sent for CBC and culturing to demonstrate the underlying pathogen behind their fever. They were 41 (70.7%) males and 17 (29.3%) females; the average age of patients was between 14 and 80 years. All of these cases suffered from hematological malignancy. Patients with hematologic malignancies with neutropenic fever were divided into four groups according to absolute neutrophil count (mild neutropenia: ANC < 1500/mm³, moderate neutropenia: ANC < 1000/mm³, severe neutropenia: ANC < 500/mm³, very severe neutropenia: ANC < 200/mm³).

Inclusion criteria in this study include all adult patients with hematological malignancy undergoing chemotherapy who presented with neutropenic fever, body temperature  of  ≥ 38.3 °C on one occasion, or ≥ 38 °C that sustained over 1 hour, with absolute neutrophil count of  <1500/mm³.

Blood collection, CBC and BacT/ Alert system

Each patient had a venous blood sample of 12 mL taken intravenously. After skin cleaning, 10ml of collected blood was utilized to inoculate the blood bottle according to usual procedure. In a BacT/ALERT blood culture system (BacT/Alert FA Plus, bioMerieux SA, France), the infected bottles of blood were incubated at 35± 2°C. Bottles of BACT/Alert that exhibited signs of growth were agitated. All samples were submitted to the Al-Sadr Teaching Hospital, Clinical and Special Laboratory right away and incubated in a BacT/Alert blood system at 35±2°C. 2 mL of the leftover blood was transferred to the CBC container, which also contained EDTA, and delivered to the hospital laboratory.

Bacterial identification and antimicrobial susceptibility testing (AST) using VITEC 2 system

To use this technique we need pure bacterial cultures. So, all affirmatively alarmed BacT/Alert system bottles were sub-cultured on 5% sheep blood culture, MacConkey agar, and Sabouraud dextrose agar (SDA) for bacterial and fungus growth, respectively, at 37°C and 30°C.

Bacterial identification and antibiotic susceptibility testing (AST) was done using automated VITEC2 system (bioMerieux, France). In order to choose suitable identification (ID) and AST cards, basic identification tests such as Gram staining, catalase and oxidase tests were done according to standard bacteriology protocol on all positive blood agar and MacConkey agar plates.

A bacterial suspension was made from each pure culture with a turbidity of 0.5 McFarland (1.5×10⁸ CFU/ml) based on VITEC2 construction. All of the prepared bacterial suspensions, as well as specific ID and AST cards, were simultaneously placed into the VITEC2 system apparatus. Following identification, the minimum inhibitory concentration (MIC) for each antibiotic was calculated. All antibiotics were chosen using the CLSI guidelines [9].

Fungi identification and antifungal susceptibility testing (AST)

Fungal identification and antibiotic susceptibility testing (AST) was done using automated VITEC2 system (bioMerieux, France). In order to choose suitable identification (ID) and AST cards. Methylene blue was used to microscopically evaluate all Sabouraud dextrose agar (SDA) plates that indicated fungal colonies.

A fungal suspension was made from each pure culture with a turbidity of (1.80-2.20) McFarland based on VITEC2 construction. All of the prepared fungal suspensions, as well as specific ID and AST cards, were simultaneously placed into the VITEC2 system apparatus. Antifungal agents including; the following treatments: amphotericin B, voriconazole, caspofungin, fluconazole, itraconazole and micafungin have been used in antifungal susceptibility testing.

Statistical Analysis

The analysis was done using the SPSS software, version 26. For descriptive purposes, frequencies and percentages were used for qualitative data and mean± standard deviation with median, minimum and maximum values were used for quantitative data. Shapiro-Willk and Kolmogorove-Smirnov tests were used to investigate quantitative data parametry. Mann Whitney U Test and Fisher's Exact Test were used to investigate the statistical significance of any difference/ association appeared in the comparisons made. 

Results

Figures & Tables

Discussion

To effectively treat infections in patients with neutropenic fever, hematologic malignancies after receiving chemotherapy, it is critical to understand potential pathogens as well as local sensitivity patterns to antibiotics and antifungals in treatment centers.

Neutropenic fever is a single oral temperature of ≥38.3º C or a temperature of ≥38.0º C sustained for more than 1 hour in a patient with neutropenia. Bloodstream infection is a major cause of increased morbidity and mortality among neutropenic patients, who have hematologic malignancies. Both bacterial and fungal germs are associated with bloodstream infection among this type of patients [7, 10]

Gram-negative bacilli were the most common causative agents of BSI in patients with cancer and neutropenia in studies conducted worldwide during the 1960s and 1970s. Gram-positive cocci (30-70 %) predominated over the next two decades [11, 14]. In our hospital, where Gram-negative bacteria predominated, this substitution was not observed. It was found that the majority of infections causing pathogens were gram-negative bacteria (15.52%), followed by gram-positive bacteria (6.89%). Escherichia coli was the predominant isolate in general (13.8%), Klebsiella pneumoniae accounted for (1.7%), Staphylococcus hominis (coagulase-negative) (5.2%), followed by Staphylococcus aureus (coagulase-positive) (1.7%).). Similar to what was found in the Iranian research; gram-negative bacteria (63.3%) were detected more frequently than gram-positive bacteria (36.7%). Escherichia coli was the most common Gram-negative organism (47%), followed by coagulase-negative Staphylococcus (CoNs) and was the most common pathogen isolated among Gram-positive bacteria (54.6%)[15]. These Escherichia coli isolates showed different antibiotic susceptibility: Imipenem (75%), Meropenem (62.5%), Amikacin (83.3%), Gentamicin (62.5%), Tobramycin (50%), Fosfomycin (100%), and Nitrofurantoin (100%). These results were close to the Iranian research, which showed additional sensitivity to Tigecycline, while in our research; this sensitivity did not appear in Escherichia coli [16].

Klebsiella pneumoniae, was found in one case in a patient suffering from aplastic anemia at the age of 17 years, suffering from severe neutropenic fever. He died due to a bloodstream infection with Klebsiella pneumoniae, which showed resistance to all kinds of drugs according to the results shown by the drug sensitivity VITEK 2 (BioMerieux) system. Klebsiella pneumoniae is one of a few bacteria that have developed a high rate of antibiotic resistance due to changes in the organism's core genome [17].

Gram-positive bacteria, Staphylococcus hominis   (coagulase-negative), and Staphylococcus aureus (coagulase-positive) have been found to cause infections in the bloodstream that can travel from the skin in their usual place to the blood in which they become pathogenic. Possible explanation may be due to the continuous use of intravenous catheters when the patient is undergoing a long course of chemotherapy or when experimental therapies are used in anticipation of possible infection in patients with neutropenia, as well as it can pass from the hands of hospital staff to the patient with neutropenia as he/ she suffers of weak immunity. This interpretation agrees with what was mentioned in a research that took place in Baghdad Teaching Hospital. Gram-positive bacteria responsible for microbiologically proven infections are responsible for 60-70% of infections. This percentage may be due to the widespread use of quinolones or the use of broad-spectrum antibiotics for empirical antimicrobial therapy for Gram-negative bacteria [18].

In our study, Staphylococcus hominis (coagulase- negative) was isolated from 3 cases. This bacterium was sensitive to the following antibiotics: Linezolid, Teicoplanin, Vancomycin, Tigecycline, Nitrofurantoin, Rifampicin, and Trimethoprim/Sulfamethoxazole.

Regarding drug sensitivity to Staphylococcus aureus (coagulase-positive), one case was sensitive to most drugs except for one treatment (Rifampicin).

This study also showed convergence in the results where, the majority of coagulase-negative staphylococci (94%) were susceptible to linezolid, and more than 80% were susceptible to (Teicoplanin, Rifampicin, Chloramphenicol and Vancomycin) [15]. A similar study was found to our results for Staphylococcus aureus , which showed sensitivity to most of the drugs used and no resistance to any treatment used (Gentamicin, Levofloxacin, Moxifloxacin, Erythromycin, Clindamycin, Vancomycin, Linezolid, Tetracycline, Tigecycline, Rifampicin, Trimethoprim/Sulfamethoxazole) [16].

Bacillus cereus, a Gram-positive facultative anaerobic bacterium, was isolated from only 1 case, a multiple myeloma patient. It is believed that infection of the bloodstream with this type of bacteria is due to the patient undergoing a long period of treatment. The infection may be from a central venous catheter or from having had abdominal surgeries, or from being infected by eating food, because immunocompromised due to chemotherapy or use much empirical antibiotics. This is consistent with research conducted in Switzerland; there were three cases of Bacillus cereus infection in the bloodstream from all 10 patients with blood diseases who had neutropenia at the time of infection. It is believed that the bloodstream sources were the intestinal tract (50%) and the central venous catheter (50%) [19, 20].

Three fungal isolates were also found in patients with neutropenic fever: Rhodotorula glutinis /mucilaginosa, Candida dubliniensis, and Candida parapsilosis.

One isolate of Rhodotorula glutinis / mucilaginosa, showed drug sensitivity to Caspofungin and Amphotericin B. This sensitivity was similar to a case report from Turkey [21].

Candida dubliniensis was isolated from a patient with acute lymphoblastic leukemia admitted to the intensive care unit suffering from neutropenic fever, and it was sensitive to Voriconazole, Amphotericin B, and Flucytosine. In a Korean study, a neutropenic man with multiple myeloma had a polymicrobial bloodstream infection with C. albicans and C. dubliniensis, from an infected central venous catheter, and these microbes were sensitive to Amphotericin B and Itraconazole [22].

Candida parapsilosis was isolated, which showed drug sensitivity to all drugs used in the test (Fluconazole, Voriconazole, Caspofungin, Micafungin, Amphotericin B, and Flucytosine). In a study conducted at the Catholic University of Rome, an increase in cases of Candida caused by C. parapsilosis was reported. It was found that candidiasis is the most common cause of candida infection in intensive care patients and patients with hematological malignancies. Drug sensitivity was tested for drugs and showed their sensitivity (Flucytosine, Fluconazole, Itraconazole, and Voriconazole) [23].

Regarding the site of infection in the scope of our study, 32.8% suffer from inflammation in the respiratory system. 22.4% have inflammation of the skin that may be in the soft tissues of the skin or ulcers in skin area. 12.1% suffer from infections, in the oropharynx. 8.6% infection in the gastrointestinal tract. 3.4% inflammation in the paranasal sinus, and 3.4% inflammation in the urinary tract.1.7% inflammation in the Perianal region, 36.2% in addition to the presence of cases suffering from neutropenic fever, but they did not show clear symptoms indicating the presence of inflammation or infection. What was found in our study was similar to what was reported by Naji, et al. in a study he conducted on neutropenic patients suffering from malignant hematomas in Baghdad Teaching Hospital, in which it was found that 52% had respiratory gas infection, 18% urinary tract infection, 16% infection in the perianal area, 12% inflammation in the sinuses, 12% inflammation in the digestive system, 12% presence of inflammation in the skin, mucous membrane and canola sites, and 2% infection of the nervous system [18,10].

Neutropenic fever is a direct cause for morbidity and mortality of patients with hematological malignancies. About 65% of our patients were culture-negative; however, the use of Meropenem or Imipenem in combination with Aminoglycoside was effective in majority of cases. Early administration of broad-spectrum antimicrobials after obtaining a sample for blood culture is crucial in way of management.

C-reactive protein is a useful biomarker for diagnosis and prognosis as it could be a predictor for mortality. 

The authors declare that there is no conflict of interest.

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