Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Background: Pontiac fever and Legionnaires' illness are both brought on by the gram-negative bacteria Legionella pneumophila (L. pneumophila). Patients with neutropenia, a typical side effect of cancer chemotherapy, may experience rapid spread of Legionella infections. By focusing on the (macrophage infectivity potentiator) mip gene, we sought to identify L. pneumophila in patients with neutropenic fever who were suffering from haematological malignancies.

Subjects and Approaches: 80 patients provided serum and urine samples, which were then examined by PCR for the presence of the L. pneumophila mip gene.

Conclusion: Based on our findings, L. pneumophila was quite common in the group of patients we analysed, demonstrating the need to take this microbe into account in future research regarding cancer patients' detection and treatment.

Keywords: Malignancy, Mip, Neutropenia, Legionella pneumophila

Legionella pneumophila (L. pneumophila) is a particular, gram-negative and facultative intracellular bacterium. It is the causative specialist of Legionnaires' sickness an extreme type of pneumoniaalso causes the Pontiac fever which incorporates a flulike, self-restricting disease. This miniature creature for the most part exists in freshwater conditions however can likewise possess in man-made water circulation frameworks,which are the fundamental wellsprings of contamination.[1] Since the first portrayal of Legionnaires' illness in 1976, L.pneumophila ontaminations have turned into a critical reason for medical clinic obtained dreariness and mortality.[2]The main methods of transmission of L.

pneumophila contaminations are through inward breath of the polluted sprayers which can follow by thereplication of microbes inside human alveolar macrophages.[3] The contamination in patients languishing from serious neutropenia can circulate quickly[4]. Next to the various reports in regards to present the gamble variables to create of L.pneumophila diseases, the extreme neutropenia which is characterized as outright neutrophil count ≤500/mm3,[5,6] and furthermore is a significant entanglement in malignant growth patients, stays as most normal inclining factor for L. pneumophila contaminations in such patients[7]. The early recognition of this disease in malignant growth patients is basic and deferring in proper treatment the mortality rate rises[8,9].

The polymerase chain reaction (PCR) has been used extensively for the detection of L. pneumophila in respiratory secretions, [10] urine, [11], serum, and environmental samples, [13], and the (macrophage infectivity potentiator) mip gene has been targeted in nucleic acid amplification-based methods for this microorganism's identification.[14,15] Despite several reports of L. pneumophila discovery in various patient populations, [15–17] there haven't been many research looking at the prevalence of this microbe in neutropenic fever patients with haematological malignancies. In order to demonstrate this, we used PCR to detect the presence of L. pneumophila in urine and serum samples from this patient group.the significance of this microorganism in such patients and the need to develop further hospital infection prevention techniques.

Tests assortment and DNA extraction Eighty patients with hospitalized hematological danger and affirmed neutropenic fever(granulocyte count of 1,000/mm3) in Imam Khomeini and Taleghani clinics, Tehran, Iran(with age range: 14-80 years of age) introduced between June 2013 to July 2014, were remembered for this review. Informed assent furthermore Data were acquired from all members.(Moral code: 89/1412 from Avicenna Exploration Foundation)Five mL fringe blood and 15 mL pee examples were acquired from every patient. The tests were put away at - 70°C until investigation. The bacterial DNA separated from serum and pee tests by Genomic DNA Extraction Pack (Bioneer,Seoul, South Korea) as indicated by make guidance.

The separated DNA from serum and pee examples were exposed to PCR of mip quality in the presence of standard PCR combination containing 20 mM Tris/HCl, pH 8.3, 100 mM KCl, 1.5 mM MgCl2,0.25 mM dNTP and 10 pM of every groundwork and 1U Taq DNA polymerase. The pre-owned groundworks in this review were as per the following: Forward preliminary succession: 5´-GCT TTA ACC GAA CAG CAA ATG-3´ and Invert groundwork

succession: 5´-AAC GGT ACC ATC AAT CAG ACG3´which made a PCR result of 267 bp. 18 The cycling states of enhancement were as per the following: starting denaturation at 95°C for 5 min followed by 37 patterns of denaturation at 95°C for 1 min, temperingat 58°C for 30 sec, augmentation at 72°C for 30 sec and last augmentation at 72°C for 5 min. The results of PCR were electrophoresed through 1.5% agarose(Promega Co., USA) gel at 80 V for 1 to 1:30 h in TAE support. Ethidium bromide 0.4 mg/ml (SigmaSynthetics Co, USA) was added to the gel to envision DNA on UV transilluminator.

Cancer patients continue to experience significant problems with infection, and neutropenic fever, a frequent side effect of anti-neoplastic chemotherapy, makes these patients more vulnerable to infections.[20,21] L. pneumophila is regarded as a significant cause of infections in cancer patients among the various bacterial agents that have been found in these patients. 7 In this investigation, we used PCR to identify the L. pneumophila mip gene in urine and serum samples from neutropenic febrile patients with haematological malignancies. In the urine and serum of 80 patients, we found the L. pneumophila mip gene in 21 (26.2%) and 38 (47.5%) cases, respectively. These results demonstrate the significance of L. pneumophila in this particular patient population and provide support for earlier research on Different indicative trategies incorporate serological testing, direct fluorescent neutralizer staining (DFA),the urinary antigen discovery and PCR-based procedures have been created for recognition of L.pneumophila diseases.

24 Discovery of immunizer levels against L. pneumophila in serum isn't appropriate conclusion strategy during the intense period of infection. DFA on respiratory examples is a quick strategy yet it has low awareness.25 Economically urinary antigen tests additionally are accessible and have been shown that can be helpful for L. pneumophila decetion.26,27 Albeit the bacterial culture remains as "highest quality level" technique for this reason, however because of the generally sluggish growing (3-10 days) and picky nature of this microorganism,24,28 moreover decline aversion to culture in examples determined from treated patients with an expansive range anti-toxin,29 different systems to guarantee a quick determination of legionellosis have become basic. The PCR-based procedures have been shown that can be promising strategy for the quick determination of L. pneumophila especially in culture and serum mimmunizer pessimistic people.30 These procedures have given adequate responsiveness,[24,31,32] thusly, we involved PCR in our review for location of this microorganism utilizing pee and serum examples. These examples have been used broadly for L. pneumophila recognition in contaminated patients.[5,33] These non-respiratory examples appear more appropriate than sputum for distinguishing proof of L.pneumophila on the grounds that it has been shown that less than half of the Legionnaires' illness patients produce sputum.

In our review, the quantities of positive cases in pee were lower than the serum tests (21 cases for pee versus 38 cases for serum from 80 patients). Albeit in ALL patients, number of positive cases for L. pneumophila by assessment of pee and serum has no very contrast (10 and 8 cases for serum and pee, separately) additionally in MM languishing people the positive cases in both pee and serum examples are equivalent however in general our discoveries

propose that pee test can't be appropriate for discovery of L. pneumophila by PCR. It would be ideal for it to be note that this discoveries should be decipher by alert since it is interestingly, for certain examinations which have been accounted for a high discovery pace of Legionella DNA in pee,[5,34,35]. In our work we didn't have bacterial culture gear to assess this contrasts among pee and serum examples in any case, basically there is one review that affirms our information about unsatisfactoriness of pee tests for recognizable proof of L. pneumophila by PCR. [36].

Albeit number of patients was not enormous and we didn't play out the bacterial culture, like other reports[37,38] yet the current information showed relative high predominance of L. pneumophila in hematological harm patients with affirmed neutropenic fever which features the significance of rethinking to forestall this sort of contaminations in malignant growth care unit climate. Moreover,gained information are interesting the PCR as appropriate also, fast strategy for recognition of L. pneumophila,anyway more investigations ought to be looked to judgment between reasonableness of pee and serum examples. Likewise, the relationship between basic issues like constant renal disappointment,hepatic cirrhosis, diabetes, coronary illness,liquor abuse, smoking and enslavement with vulnerability to L. pneumophila disease in these patients ought to be assessed in ongoing examinations.

Our results showed a very high incidence of L. pneumophila in neutropenic febrile patients with haematological malignancies, demonstrating the need to take this microbe into account in future studies from the perspective of cancer patients' identification and treatment.

1. Fraser DW, Tsai TR, Orenstein W, et al. Legionnaires'disease: description of an epidemic of pneumonia. N EnglJ Med. 1977; 297(22):1189-97.

2. Oren I, Zuckerman T, Avivi I, et al. Nosocomial outbreak of Legionella pneumophila serogroup 3 pneumonia in a new bone marrow transplant unit: evaluation, treatment and control. Bone Marrow Transplant. 2002; 30(3):175-9.

3. Albert-Weissenberger C, Cazalet C, Buchrieser C.Legionella pneumophila- a human pathogen that coevolved with fresh water protozoa. Cell Mol Life Sci.2007;64(4):432-48.

4. Bodey GP, Buckley M, Sathe YS, et al. Quantitative relationships between circulating leukocytes and infection in patients with acute leukemia. Ann Intern Med. 1966; 64(2):328-40.

5. Murdoch DR, Walford EJ, Jennings LC, et al. Use of the polymerase chain reaction to detect Legionella DNA in urine and serum samples from patients with pneumonia. Clin Infect Dis. 1996; 23(3):475-80.

6. Hughes WT, Armstrong D, Bodey GP, al e. 2002 Guidelines for the use of antimicrobial agents in neutropenic patients with cancer. Clin Infect Dis.2002(34):730-51.

7. Jacobson KL, Miceli MH, Tarrand JJ, et al. Legionella pneumonia in cancer patients. Medicine (Baltimore).2008; 87(3):152-9

8. Heath CH, Grove DL, Looke DF. Delay in appropriate therapy ofLegionella pneumonia associated with increased mortality. Eur J Clin Microbiol Infect Dis. 1996;15(4):286-90.

9. Gacouin A, Le Tulzo Y, Lavoue S, et al. Severe pneumonia due to Legionella pneumophila: prognostic factors, impact of delayed appropriate antimicrobial therapy. Intensive Care Med. 2002; 28(6):686-91.

10. Cloud JL, Carroll KC, Pixton P, et al. Detection of Legionella species in respiratory specimens using PCR with sequencing confirmation. J Clin Microbiol. 2000;38(5):1709-12.

11. Maiwald M, Schill M, Stockinger C, et al. Detection of Legionella DNA in human and guinea pig urine samples by the polymerase chain reaction Eur J Clin Microbiol Infect Dis. 1995; 14(1):25-33.

12. Murdoch DR, Chambers ST. Detection of Legionella DNA in peripheral leukocytes, serum, and urine from a patient with pneumonia caused by Legionella dumoffii. Clin Infect Dis. 2000; 30(2):382-3.

13. Delgado-Viscogliosi P, Solignac L, Delattre JM.Viability PCR, a culture-independent method for rapid and selective quantification of viable Legionella pneumophila cells in environmental water samples. Appl Environ Microbiol 2009;75(11):3502-12.

14. Tachibana M, Nakamoto M, Kimura Y, et al.Characterization of Legionella pneumophila Isolated from Environmental Water and Ashiyu Foot Spa. Biomed Res Int. 2013;2013:514395.

15. Ghotaslou R, Yeganeh Sefidan F, Akhi MT, et al.Detection of legionella contamination in tabriz hospitals by PCR assay. Adv Pharm Bull. 2013; 3(1):131-4.

16. Kaku N, Yanagihara K, Morinaga Y, et al. Detection of Legionella pneumophila serogroup 1 in blood cultures from a patient treated with tumor necrosis factor-alpha inhibitor. J Infect Chemother. 2013; 19(1):166-70.

17. Arinuma Y, Nogi S, Ishikawa Y, et al. Fatal Complication of Legionella pneumophila Pneumonia in a Tocilizumab-treated Rheumatoid Arthritis Patient. Intern Med. 2015; 54(9):1125-30.

18. Gilmour MW, Bernard K, Tracz DM, et al. Molecular typing of a Legionella pneumophila outbreak in Ontario,Canada. J Med Microbiol. 2007; 56(Pt 3):336-41.

19. Schürmann D, Ruf B, Pfannkuch F, et al. Fatal legionellosis in patients with malignant hematologic diseases. Blut. 1988;56(1):27-31.

20. Nesher L, Rolston KV. The current spectrum of infection in cancer patients with chemotherapy related neutropenia. Infection. 2014; 42(1):5-13.

21. Bow EJ. Infection in neutropenic patients with cancer.Crit Care Clin. 2013; 29(3):411-41.

22. Kosmidis CI, Chandrasekar PH. Management of grampositive bacterial infections in patients with cancer. Leuk Lymphoma. 2012; 53(1):8-18.

23. Gudiol C, Verdaguer R, Angeles Domínguez M, et al.Outbreak of Legionnaires' disease in immunosuppressed patients at a cancer centre: usefulness of universal urine antigen testing and early levofloxacin therapy. ClinMicrobiol Infect. 2007; 13(11):1125-8.

24. Murdoch DR. Diagnosis of Legionella infection. ClinInfect Diseases. 2003;36: 64–9.

25. Helbig JH, Uldum SA, Luck PC, et al. Detection of Legionella pneumophila antigen in urine samples by the BinaxNOW immunochromatographic assay and comparison with both Binax Legionella Urinary Enzyme Immunoassay (EIA) and Biotest Legionella Urin Antigen EIA. J Med Microbiol. 2001; 50(6):509-16.

26. Ramirez JA, Summersgill JT. Rapid tests for the diagnosis of Legionella infections. J Ky Med Assoc. 1994;92(2):62-5.

27. Benson RF, Tang PW, Fields BS. Evaluation of the Binax and Biotest urinary antigen kits for detection of Legionnaires' disease due to multiple serogroups and species of Legionella. J Clin Microbiol. 2000; 38(7):2763-5.

28. Den Boer JW, Yzerman EP. Diagnosis of Legionella infection in Legionnaires’ disease. Eur J Clin Microbiol Infect Dis. 2004; 23(12):871-8.

29. Mercante JW, Winchell JM. Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations. Clin Microbiol Rev. 2015; 28(1):95-133.

30. Koide M, Higa F, Tateyama M, et al. Detection of Legionella species in clinical samples: comparison of polymerase chain reaction and urinary antigen detection kits. Infection. 2006; 34(5):264-8.

31. Jaulhac B, Nowicki M, Bornstein N, et al. Detection of Legionella spp. in bronchoalveolar lavage fluids by DNA amplification. J Clinical Microbiol. 1992; 30(4):920-4.

32. Cloud J, Carroll K, Pixton P, et al. Detection of Legionella species in respiratory specimens using PCR with sequencing confirmation. J Clinical Microbiol. 2000;38(5):1709-12.

33. Jarraud S, Descours G, Ginevra C, et al. Identification of Legionella in Clinical Samples. Methods Mol Biol. 2013;954:27-56

34. Helbig J, Engelstädter T, Maiwald M, et al. Diagnostic relevance of the detection of Legionella DNA in urine samples by the polymerase chain reaction. Eur J ClinMicrobiol Infect Dis. 1999; 18(10):716-22.

35. Matsiota‐Bernard P, Waser S, Vrioni G. Detection of Legionella pneumophila DNA in urine and serum samples from patients with pneumonia. Clin Microbiol Infect. 2000; 6(4):223-5.

36. Mentasti M, Fry NK, Afshar B, et al. Application of Legionella pneumophila-specific quantitative real-time PCR combined with direct amplification and sequencebased typing in the diagnosis and epidemiological investigation of Legionnaires’ disease. Eur J Clin MicrobiolInfect Dis. 2012; 31(8):2017-28.

37. Del Castillo M, Lucca A, Plodkowski A, et al. Atypical presentation of Legionella pneumonia among patients with underlying cancer: A fifteen-year review. J Infect.2016; 72(1):45-51.

38. Han XY, Ihegword A, Evans SE, et al. Microbiological and Clinical Studies of Legionellosis in 33 Patients with Cancer. J Clinical Microbiol. 2015; 53:2180-87.

Omid Zarei .Urine and serum samples from neutropenic febrile patients with haematological malignancies were found to have Legionella pneumophila. World Journal Of Hematology And Oncology 2022.