IMMUNE-MECHANISM, SEROLOGICAL AND HEMATOLOGICAL FINDING OF TYPHOID FEVER dr. Andi Meutiah Ilhamjaya INTRODUCTION Definition Typhoid fever is an acute systemic infectious disease caused by the salmonella enterica serovar typhi microorganism known as salmonella typhi. Salmonella is an intracellular, rod-shaped, flagellated, facultative anaerobic gram-negative bacteria that infects humans by contaminating food and water.1-3 Salmonella typhi bacteria consist of 3 types of antigen structures, namely O antigen (somatic antigen), H antigen (flagella antigen) on flagella and fimbria (pili), and Vi antigen (surface) on the capsule.3-4 Epidemiology Studies of the disease worldwide estimate that 14.3 million cases of typhoid fever occur each year, and the case fatality rate has fallen to <1% since the antibiotic era ushered in a set of highly effective treatment options. However, In recent years there have been several changes in the epidemiological pattern of typhoids in Africa, Asia and Latin America. In Indonesia, typhoid fever is endemic with an increasing trend from year to year with an average morbidity of 500 / 100,000 population and mortality rates. between 0.6 - 5%.5-6 Transmission Humans are the only reservoir for this infection. The disease is transmitted via the faecal-oral route through contaminated food and water. It is usually caused by consuming impurified water and contaminated food. As S. typhi bacteria can survive in water for days, contamination of surface water such as sewage, fresh water and ground water acts as major aetiological agent of typhoid. Defaecation in open places is another notable cause of typhoid transmission. Amidst food, cut fruits kept uncovered for some time are an important cause of contamination in most developing countries. Papaya has a neutral pH and its cut surface can support the growth of various microorganisms. It was observed by Hosoglu et al in a Turkish study that eating cut papaya, lettuce salad and some traditional raw foods in Turkey (e.g. cig kofte) was an important causative factor. Inhabiting in a congested locality or household is significantly related with typhoid fever. Again, the habit of washing vegetables and compulsory use of sanitary latrine for defecation have been found to prevent typhoid. In a case-control study in Indonesia, paratyphoid fever was found to be associated with consumption of food from street vendors.6-7 Clinical Findings Following the incubation period of 7 to 14 days, there is onset of fever and malaise. The fever is fluctuating, especially in the evening and at night with an intermittent pattern and step ladder temperature rises. A persistent high fever can also be found until the second week, accompanied by constipation, headache, abdominal pain, coughing, and vomiting. A serious complication of typhoid fever is intestinal bleeding, sometimes accompanied by perforation, and typhoid encephalopathy. 8-9 PATHOGENESIS OF TYPHOID FEVER IMMUNE MECHANISM OF TYPHOID FEVER In healthy individuals, the host body can recognize and clear pathogens through the innate and acquired immunity by a strong host immune response. However, invasive Salmonella can evade the immune surveillance using the sophisticated strategies, and could replicate, survive, and cause the persistent bacterial infections in hosts without even exhibiting the typical clinical symptoms. For example, it has been reported that, in certain cases, patients with typhoid fever may carry bacteria in their gallbladder for the rest of their lives. In general, such infections do not show clinical symptoms, but are a potential threat to the host. These asymptomatic carriers presumably act as reservoirs for a diverse range of S. Typhi strains and may act as a breeding ground for new genotypes. It has been reported that S. Typhi chronic infection facilitates the gallbladder cancer development in humans. S. Typhimurium involved in the persistent infections is also difficult to eliminate, and infected patients often continue shedding these pathogens in the environment, resulting in disease transmission 10 Typhoid is a systemic disease that varies in severity. Recently a novel model has been reported that allows analyses of the pathogenesis of S. Typhi in a humanized non-obese diabetic (NOD) severe combined immune deficient (SCID) mouse model. The understanding oftyphoid fever pathogenesis, especially the cellular and molecular phenomena that are responsible for clinicalmanifestations ofthis disease, has greatly increased with several important discoveries. These include: (a) Bacterial type III protein secretion system. (b) The virulence genes of Salmonella spp. encoding five different Sips (Salmonella invasion protein) namely Sip A, B, C, D and E, which are capable of inducing apoptosis in macrophages. (c) The function of Toll R2 and Toll R4 receptors present in the macrophage surface (originally discovered in Drosophila). The Toll family receptors are critical in cell signaling mediated through macrophages in association with lipopolysaccharidebinding protein (LBP) and CD14. (d) The lines of immune defense between intestinal lumen and internal organs. (e) The fundamental role of the endothelial cells in inflammatory deviation from bloodstream into tissues infected by bacteria.10 2.1. Intestinal mucosal immunity (first line of defense) The infectious dose of S. Typhi in volunteers varies between 1000 and 1 million organisms. The low gastric pH is an important defense mechanism as the bacteria must survive the gastric acid barrier to reach the small intestine. In the small intestine, bacteria move across the intestinal epithelial cell (CEI) and reach the M cells, thus penetrating in the Peyer’s patches (Fig. 1). The M cells are specialized epithelial cells overlying Peyer’s patches that have probably originated from CEI and small pockets in the mucosal surface. After contact with M cells, the infectious bacteria are rapidly internalized and they reach a group of antigen-presenting cells (APCs), being partially phagocytized and neutralized. The infected phagocytes are organized in discrete foci that become pathological lesions, surrounded by normal tissue. Lesion formation is a dynamic process that requires the presence of adhesion molecules such as ICAM1 (Inter-Cellular Adhesion Molecule 1),VCAM-1 (Vascular CellAdhesion Molecule 1) and the balanced action of cytokines [tumor necrosis factor (TNF)-Alpha , interleukin (IL)-12, IL-18, IL-14, IL-15 and interferon (IFN)-Gamma]. Failure to form pathological lesions results in abnormal growth and dissemination of the bacteria in the infected tissue. Some bacteria escape this barrier, and reach the developed lymphoid follicles (Peyer’s patches); formed mainly by mononuclear cells as T lymphocytes, as well as dendritic cells (DC). DC presents the bacterial antigens to immune cells that provoke activation of T and B lymphocytes. 2.2. Dissemination from intestinal mucosa’s lamina propria The T and B lymphocytes come out from the lymphatic nodules and reach liver and spleen via reticuloendothelial system (Fig. 1). In these organs the bacteria are killed mainly by phagocytosis through the macrophage system. However, Salmonella are able to survive and multiply within the mononuclear phagocytic cells (House et al. 2001). At a threshold level, determined by the number of bacteria, the bacterial virulence and the host immune response, the bacteria are released from their sequestered intracellular habitat into the bloodstream. This bacteremic phase of disease is characterized by dissemination of the organisms. The most common sites of secondary infection are the liver, spleen, bone marrow, gallbladder and Peyer’s patches in the terminal ileum. In liver, S. Typhi provokes Kupffer cell activation. Kupffer cells have high microbicidal power and neutralize the bacteria with oxidative free radicals, nitric oxide as well as enzymes, active in acid pH. The survived bacteria invade hepatocytes and cause cellular death, mainly by apoptosis. 3. Host immune defense The main host defense against Salmonella spp. occurs through the neutrophils, followed by mononuclear cells. These inflammatory cells produce cytokines as TNF-Alpha, IFN-Gamma, IL-1, IL-2, IL-6 and IL-8. The Kupffer cells are the main TNF- producer in the liver. Clearance of bacteria from tissues requires the CD28-dependent activation of CD4+, T cell receptor (TCR) - alpha beta T cells and is controlled by Major histocompatibility complex (MHC) class II genes. DC and B-cells are involved in the initiation and development of T-cell immunity to Salmonella. Interaction between B and T-cells is needed for the development of antibody response to Salmonella proteins and for isotype switching of antibody response against lipopolysaccharide antigens. Resistance to reinfection with virulent Salmonella microorganisms (secondary infection) in immunized mice requires the presence of CD4+ dependent Th1 type immunological memory, CD8+ T cells and anti-Salmonella antibodies. The development of Salmonella-specific CD4 effector responses has been examined in both susceptible and resistant mice. Fig. 1. Schematic representation of persistent infection with Salmonella enterica serovar Typhi in humans: bacteria enter the Peyer’s patches of the intestinal tract mucosal surface by invading M cells – specialized epithelial cells thattake up and transcytose luminal antigens for uptake by phagocytic immune cells. This is followed by inflammation and phagocytosis of bacteria by neutrophils and macrophages and recruitment of T and B cells. In systemic salmonellosis, such as typhoid fever, Salmonella may target specific types of host cells, such as dendritic cells and/or macrophages that favour dissemination through the lymphatics and blood stream to the mesenteric lymph nodes (MLNs) and to deeper tissues. This then leads to transport to the spleen, bone marrow, liver and gall bladder. Bacteria can persist in the MLNs, bone marrow and gall bladder for life, and periodic reseeding of the mucosal surface via the bile ducts and/or the MLNs of the small intestine occur, and shedding can take place from the mucosal surface. Interferon-(IFN-), which can be secreted by T cells, has a role in maintaining persistence by controlling intracellular Salmonella replication. Interleukin (IL)-12, which can increase IFNproduction and the proinflammatory cytokine tumor-necrosis factor-(TNF-α) also contribute to the control of persistent Salmonella. These studies suggest massive expansion of Salmonella-specific CD4 T cells and rapid acquisition of Th1 effector functions, namely the enhanced ability to secrete TNF-Alpha, IFN-Gamma and IL-2 upon restimulation. Epithelial cells seem to play the central role in coordinating the inflammatory response to intestinal pathogens. The interaction of Salmonella spp. with epithelial cells leads to the generation of a great number of biochemical signals by these cells. These include the basolateral release of chemokines (including IL-8) and apical secretion of “pathogen-elicited epithelial chemoattractant” (PEEC). These substances are partially responsible for guiding the recruitment and traffic of PMNs (Polymorphonuclear Leukocytes) across CEIs.After initial localization in resident phagocytes (macrophages) the bacteria associate mainly with PMNs in early phase of infection. It was therefore reasonable to assume that PMNs control early bacterial growth in tissue. However, evidence for a predominant role of mononuclear cells and not the PMNs in early resistance to the disease has been provided. In a study S. Typhimurium (the rodent counterpart of S. Typhi that causes salmonellosis in mice/rats with typhoid like symptoms) infection was shown to induce IL-8 secretion by intestinal epithelium that was mediated through increase in intracellular calcium. This phenomenon was found to be NF-KB dependent. A functional T3SS (type III secretion systems) is required for the induction of PMN transmigration. Furthermore, protein synthesis in both bacteria and epithelial cells is also required for this activity. Invasion of Salmonella spp. into epithelial cells is insufficient for trans-epithelial signaling to PMNs, and PMN migration occurs even when Salmonella spp. invasion is blocked. The ‘inflammatory deviation’ that happens when blood leukocytes migrate across endothelial cells into hepatic and spleen tissues is another important event. This phenomenon occurs through the action of adhesion molecules named integrins (chain ) in inflammatory cells and selectins in endothelial cells (E and P). Afterward, selectins are substituted by ICAM and VCAM proteins (whose partner in inflammatory cells is VLA4 or 4 1 integrin). The inflammatory microenvironment is completed by chemokines that are capable of stimulating leukocyte motility (chemokinesis) and directed movement (chemotaxis) of neutrophils and mononuclear cells. Chemokines bind to CC and CXC receptors in the surface of inflammatory cells. The chemokines help the blood leukocyte migration directly to host cells infected by bacteria. TNF- is produced by macrophages and other mononuclear cells and has much antibacterial activity against Salmonella spp. Besides the macrophage phagocytosis, TNF- in association with IFN-, IL-2 and other cytokines, is responsible for the neutralization of these invasive bacteria. Bacteria infested Peyer’s patches produce strong inflammatory reaction with the recruitment of leukocytes. The potent inflammatory reaction against Salmonella species provokes host cell death, as well as apoptosis of both inflammatory and epithelial cells following nutrient deprivation and termination of bacterial replication. The inflammatory response of the Th1-dominant type is destructive for host cells and for bacteria; it attenuates progressively and coincides with increase of the Th2-immune response. Th2 cells produce IL-4, IL-10, IL-13 and transforming growth factor (TGF) that cause powerful protective effect on host cells (hepatocytes, CEIs, inflammatory cells, etc.) through partial inhibition of cytokines associated with the Th1 response. Salmonella flagella have been implicated in host early innate immunity against Salmonella as these cause intestinal epithelial or macrophage inflammation following infection. S. Typhi flagella induced cytokine release from human monocytes and impaired antigen presentation by human macrophages. Flagellin of Salmonella suppresses epithelial apoptosis and limits disease during enteric infection (Fig. 2). It has been demonstrated that flagellin was the componentthat activated Toll-like receptor 5 (TLR). Moreover, studies have identified Intracellular IL-1-converting enzyme protease-activating factor (Ipaf) as an essential sensor for cytoplasmic flagellin, which activates caspase-1 and induces the production of the proinflammatory cytokine IL-18. Using TLR5-deficient mice showed that while TLR5 was crucial for the in vivo recognition of flagellin, it may also participate in the detection of systemic infection by S. Typhimurium. TLR5 activates NF-KB and the mitogen-activated protein kinases (MAPKs)leading to the secretion ofmany cytokines, including IL-6, IL-12, and TNF-Alpha, whereas Ipaf permits the activation of caspase-1 and secretion of mature IL-1. The activationofmacrophages by Lipopolysaccharide (LPS)from Salmonella species also results in the release of a variety of inflammatory cytokines, such as IL-6 and IFNGamma, which were not detected in macrophages of TLR4 knockout mice. Following binding to LPS, in association with proteins MD2 and CD14, TLR4 dimerizes and undergoes a conformational change required for the recruitment of downstream Toll/interleukin-1 receptor (TIR) domaincontaining adaptor molecules to activate both NF-kB and MAPKs. Further, TLR4 triggers the early response to Salmonella and TLR4 and TLR2 are required sequentially for efficient macrophage function in Salmonella infections. TLR2 canrecognize Salmonella lipoproteins and lipoteichoic acid, probably in cooperation with TLR6 and/or TLR1. TLR9 is activated by bacterial DNA (detecting unmethylated Cp motifs). TLR1, TLR2, and TLR9 are up-regulated while TLR6 is down-regulated which accounts for the plateau phase observed during sublethal S. Typhimurium infection in rodents. These results suggest that in addition to TLR4, the TLR2-TLR1 complex and TLR9 may play a role in controlling infection, particularly in the later stages when the bacterial growth is suppressed, possibly at the adaptive phase of the immune response. LABORATORY DIAGNOSIS FOR TYPHOID FEVER Diagnosis cannot be made on clinical grounds alone, although the presence of rose spots in a febrile patient is highly suggestive. Samples of blood, faeces and urine should be cultured on selective media. An antibody response to infection can be detected by an agglutination test (Widal test), but non-specific cross-reaction with other enterobacteria may also cause an increase in H and O antibody levels. Interpretation of the results is complicated and depends on knowing the normal antibody titres in the population and whether the patient has been vaccinated. A demonstration of a rising titre between acute- and convalescent-phase sera is more useful than examination of a single sample. At best, the results confirm the microbiological diagnosis; at worst they are misleading.11 1. Isolation of organism a. Blood culture This is the standard diagnostic method; it is positive in 60 to 80 percent of patients with typhoid. Culture of the bone marrow is more sensitive, around 80 to 95 percent patients, even in patients taking antibiotic for several days, regardless of the duration of illness. Blood culture is less sensitive than bone marrow because there is lower number of organism in blood than bone marrow. The sensitivity of blood culture is higher in the first week of illness, increases with the volume of blood cultured (10-15ml should be taken from school-children and adults, 2-4ml are required from toddlers and preschool children). Toddlers have higher level of bacteraemia than adult.12 b. Stool Culture The sensitivity of stool culture depends on the amount of faeces cultured, and the positivity rate increased with the duration of illness. Stool cultures are positive in 30 percent of patients with acute typhoid fever.12 c. Urine Culture Urine culture have got 0-58% sensitivity.12 2. Serology and haematology test a. The tubex test The samples are contained in different chambers in the reaction vessel, placed on a magnet-embedded stand. Also shown is the color-standard chart. 12 Procedure: Basically, a drop (25 Al) of reagent A (LPS-sensitized magnetic particles) was placed in a chamber of the reaction vessel. A drop of the unknown serum or antibody preparation was then added to the chamber and the contents mixed rapidly for 1 min. Next, two drops of reagent B (IgM mAbsensitized indicator particles) were added and the contents were mixed again rapidly (1 min) and thoroughly. The result was read after standing the reaction vessel on the magnetic stand, and scored according to the color chart provided. When the 3- or 6-Am non-magnetic microspheres were used instead of magnetic particles as the antigen adsorbent in some experiments, the reaction mixture was allowed to stand at room temperature for 30 min to enable all the antigensensitized particles to sediment by gravity. The method used for antigen coating is passive adsorption, as described previously.13 Result: Reading of TUBEX® TF test results done after 5 minutes of the sedimentation process magnetic particles with a magnet found on a magnetic support. Results (semiquantitative) are read in a Pad sample Filter Pad conjugate Membrane nitrocellulose Plastic backing card visually based on the color that looks after the mixing reaction is carried out and compared to the existing color scale on the TUBEX® TF kit, the result score ranges are of 0 (red color, very negative) megatin up to 10 (dark blue, very positive color).13 Performa: Tubex has not been evaluated extensively but in preliminary studies, this test performed better than Widal test in both sensitivity and specificity.12 Among the advantages of the TUBEX® test TF is: (1) has sensitivity and relatively high specificity, (2) using LPS O9 antigen Salmonella enterica serovar Typhi very specific, (3) procedure very easy checks so it can be performed by technicians without special training, (4) can be done anywhere, not necessarily on in the laboratory, (5) can test a lot test at once so it can be used on mass screening, (6) results can be obtained online fast approximately 10 minutes, (7) blood samples it takes only a little, noninvasive.13 b. Typhidot :12.13 - Can detect IgM and IgG antibodies against 50 kD antigen of Salmonella typhi - Can performed in the first 4-5 days of fever Procedure: Patient sample (serum / plasma / blood intact) dropped on the sample pad (Figure 2.8), for whole blood samples, bufer should be dropped on the sample pad after the patient sample.13 Absorbent pad at end causes capillaries, which fit the sample (and support) against the filter. When the sample flow through the filter, red blood cells in the whole blood sample contained in the filter, while serum containing antibodies the patient passes through the filter towards the conjugate pad.13 Although culture remains gold standard, Typhidot-M is superior to culture method in sensitivity (93%) and has high negative predictive value.12 3. Titration of antibody against S.typhi a. Widal Test Widal test is the investigation of choice second week and third week. As antibodies appear only after the end of the first week, it is not preferred in first week of illness.14 The classic Widal test is more than 100 years old. It detects agglutinating antibodies to the O and H antigens of S. enterica serotype typhi. The levels are measured by using doubling dilutions of sera in large test tube.54 Although easy to perform, this test has moderate sensitivity and specificity.58 Its reported sensitivity is 70 to 80 percent with specificity 80 to 95 percent.12 Principle: It is an agglutination test where H and O antibodies are detected in the patient’s sera by using H and O Antigen.14 Results: 1. O agglutination appears as compact granular chalky clumps (disc-like pattern), with clear supernatant fluid. 2. H agglutination appears as large loose fluffy cotton-woolly clumps, with clear supernatant fluid. 3. If agglutination does not occur, button formation occurs due to deposition of antigens and the supernatant fluid remains hazy. 4. Antibody Titer: The highest dilution of sera, at which agglutination occurs.14 False positive Widal test may occur due to: ○ Anamnestic response: It refers to a transient rise of titer due to unrelated infections (malaria, dengue) in persons who have had prior infection or immunization. ○ If bacterial antigen suspensions are not free from fimbriae ○ Persons with inapparent infection or prior immunization (with TAB vaccine) 14 Four-fold rise in antibody titer in paired sera at 1week interval is more meaningful than a single high titer: ○ After 1 week if titer rises-indicates true infection ○ After 1 week if titer falls- indicates anamnestic responses14 False negative Widal test may occur in: ○ Early stage (1st week of illness), Late stage (after fourth week) and in carriers ○ Patients on antibiotics ○ Due to prozone phenomena (antibody excess): This can be obviated by serial dilution of sera. 14 The interpretation of the results is positive if the agglutinin O titer is at least 1/320 or there is an increase in the titer up to four times on reexamination with an interval of 5-7 days.13 SEROLOGICAL FINDINGS AND BASIC MECHANISM Tubex Findings Tubex can only detect salmonella typhi IgM antibodies. Can be performed in the first 4-5 days of fever.12,13 Basic mechanism TUBEX TF test using the method Magnetic Binding Inhibition Immunoassay (IMBI®) to detect serum antibodies specific (IgM) against the O9 antigen found in the Salmonella lipopolysaccharide (LPS) enterica serovar Typhi. O9 antigen can be immunodominant stimulates the immune response independently, this antigen can directly stimulate mitosis B cells without the need for help from T cells. Because it has this property, it has an immune response against the O9 antigen is fast, so that detection of anti O9 antibodies can done earlier, namely on the 5th day for primary indication and 2nd day for infection secondary.13 Immunoglobulin M (IgM) can detected the ability to inhibits the adhesion reaction between the reagentse monoclonal antibody (anti-O9 mAb) labelled blue latex with O9 antigen reagent LPS labeled Salmonella enterica serovar Typhi magnetic latex particles, which bond inhibition will later be separated by a magnetic power. The components that play a role in the method These IMBIs are: (i) LPS O9 antigen particles Salmonella enterica serovar Typhi labelled magnetic latex (Chocolate reagent), (ii) Particles anti-O9 labeled monoclonal antibody colored latex particles (blue reagent), (iii) Magnetic stand serves to settle the attachment antigenantibody particle bonding.13 Figure 3. This figure illustrating how TUBEX works for detecting anti-O9 antibodies or for detecting O9 antigen. Shown are the antibody-bound indicator particles and the antigen-bound magnetic particles that are inhibited from binding to each other by the patient’s antibodies or antigen.15 Typhidot Findings Typhidot can detect IgM and IgG antibodies against 50 kD antigen of Salmonella typhi and can performed in the first 4-5 days of fever.12,13 Basic mechanism The Typhidot is a test solid-phase immunochromatography is indirect. Salmonella typhi specific antigen moves to cellulose nitrate membrane strips. When the test sample added to the sample pad, will migrate to on. If specific antibodies are in the sample test (serum or plasma), will form antigen-antibody complex with antigen move in the test window zone. Antigen-antibody the bound complex is then detected by dyes conjugated IgM goat anti human when chese buf er is added and migrated downwards, giving it a purplish pink color.13 The control line contains the rabbit anti-goat IgG bind with the conjugated goat anti dyes human IgM. The control band serves as plus indications of proper migration with control reagents.13 Figure 4. This figure illustrating how Typhidot works for detectingSalmonella typhi.13 Widal Test Findings It can be negative in up to 30% of culture proven typhoid fever, because of blunted antibody response by prior use of antibiotic. Moreover, patients with typhoid may show no detectable antibody response or have no demonstrable rise in antibody titre. Unfortunately, S. enterica serotype typhi shares these antigens with other salmonella serotypes and shares these cross-reacting epitopes with other Enterobacteriaceae. This can lead to false positive results. If paired serums are available a fourfold rise in the antibody titre between convalescent and acute sera is diagnostic. Considering the low cost of Widal test, it is likely to be the test of choice in many developing countries. This is acceptable, as long as the results of the test are interpreted with care, on the background of prior history of typhoid, and in accordance to appropriate local cut-off values for the determination of positivity.12 HEMATOLOGICAL FINDINGS AND BASIC MECHANISM Routine haematology with leucocyte count can show: leucopenia or leucocytosis, or normal leucocyte count, relative lymphocytosis, monocytosis, mild thrombocytopenia, anemia.12 Fifteen to 25% patients show leucopoenia and neutropenia. Leucocytosis found in intestinal perforation and secondary infection. In younger children, leucocytosis is common association and may reach 20,000-25,000/mm3. 12 SUMMARY 1. Typhoid fever is an fecal-oral transmitted infectious disease →by Salmonella Typhi via consuming impurified water and contaminated food. 2. Salmonella typhi consist of 3 types of antigen structures: O antigen, H, and Vi → infection 3. Polysaccharide capsule Vi has a protective effect against the bactericidal action on the serum of infected person. 4. Infective dose of Salmonella: Minimum 10 3 –106 bacilli are needed. 5. S.typhi can escape to innate and adaptive immune system → causing many clinical findings. 6. High gastric acidity is one important barrier against invasion of S. typhi and a low gastric pH is therefore an important defence mechanism. 7. Bacterial culture is gold standard, widal test is not recommended any more by WHO. REFERENCE 1. De Abrew Abeysundara, P.; Dhowlaghar, N.; Nannapaneni, R.; Schilling, M.W.; Mahmoud, B.; Sharma, C.S.; Ma, D.P. Salmonella enterica growth and biofilm formation in flesh and peel cantaloupe extracts on four food-contact surfaces. Int. J. 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