INTRODUCTION
Undifferentiated acute febrile illness (AFI) is a common clinical syndrome among patients seeking hospital care in Egypt. Recent findings show that 5% of these patients are culture-positive for Salmonella enterica serogroup Typhi, 3% for Brucella spp., and 2% for other pathogens. An additional 18% of undifferentiated AFI patients show positive serology for typhoid fever and 11% for brucellosis.1,2 A recent retrospective study conducted at the U.S. Naval Medical Research Unit No. 3 (NAMRU-3) on Egyptian patients’ sera showed that 16% of the unexplained (i.e., non-typhoid fever/brucellosis) AFI cases were seroreactive to Leptospira antigen by IgM-ELISA and microscopic agglutination test (MAT).3 The occurrence of multiple agent infections among Egyptian AFI patients is unknown. Coinfections of malaria with leptospirosis,4 filariasis,5 HIV,6 Hantavirus,7 and others have previously been reported in Thailand and Indonesia. The purpose of the present report was to look for potential coinfections among undifferentiated AFI patients from Egypt, who participated in the ongoing surveillance from June 2005 to August 2006.
This study protocol was approved by the NAMRU-3 Institutional Review Board (IRB) (Protocol DoD Assurance 30969; NAMRU-3 CPHS 079) and Egyptian health authorities in compliance with all Federal regulations governing the protection of human subjects.
MATERIALS AND METHODS
Four select infectious-disease hospitals located in urban and/or rural areas of Egypt with upgraded laboratory capacity to cover the diagnosis of typhoid fever, brucellosis, leptospirosis, and rickettsial infections participated in this study. Surveillance sites varied from overcrowded urban hospitals to those that serve dense agricultural areas. In compliance with The World Health Organization (WHO) case definition criteria, a case of acute febrile illness (AFI) was defined as any individual with fever for at least 2 days or temperature on admission of 38.5°C or greater; age ≥ 4 years with no identified cause of fever, such as diarrhea or pneumonia; or suspected of having typhoid fever or brucellosis, as defined by WHO.8 Informed consent was obtained from all adult participants and from parents or legal guardians of minors prior to participation in the study.
Blood samples and demographic characteristics and risk factors—including occupation, water, and animal or insect contact—were obtained from AFI patients upon enrolling in the study. Blood was screened for evidence of typhoid fever, brucellosis, leptospirosis, and rickettsiosis by the presence of elevated antibody levels in acute and convalescent serum specimens and/or culture (BACTEC; BD Diagnostics, MD) at the time of admission to the infectious-disease hospital. For leptospiral cultures, we used Ellinghausen–McCullough–Johnson–Harris (EMJH) media inoculated with 1–3 drops of patient’s blood incubated at 30°C and monitored weekly for 2–3 months using dark-field microscopy.9 A serological-confirmed case of typhoid fever and brucellosis was defined as a 4-fold rise between acute and convalescent serology (Brucella or Typhoid tube agglutination and/or Brucella ELISA) as previously described.1,10 When single acute serum samples were used in a subset of patients who did not donate convalescent sera, cutoff values for antibody titers, for both typhoid fever and brucellosis, were ≥ 320 units, a value that, along with relevant clinical signs, has been designated by WHO to confirm the diagnosis of these diseases in endemic areas.8 Laboratory criteria for a confirmed diagnosis of leptospirosis were the isolation of Leptospira in culture, a 4-fold or greater rise in Leptospira microagglutination titer between paired sera,3 or by PCR using ligA primers.11 A confirmed case of rickettsioses was defined as a 4-fold rise in titer between acute and convalescent sera by Typhus group Rickettsiae (TGR) IgG-ELISA,12 and/or quantitative real-time PCR (17-kDa primers).13 For TGR ELISA, the values recommended for positive reactions were used taking in consideration that background or cross-reactive antibodies are generally low-level and have been eliminated by the use of relatively higher cutoff readings (cutoff value ≥ 0.2 TGR ELISA).
RESULTS
Of 1,510 patients with undifferentiated AFI enrolled in the study to date, 187 (12.4%) showed laboratory evidence for combined infections. The rest of the patients demonstrated single AFI infections near previously reported rates.3 Of 187 patients, 20 (10.7%) were conclusively confirmed to have serial, dual, or concurrent acute infections by gold-standard laboratory tests; culture positivity and/or rising antibody titers (≥ 4-fold increase) or positive PCR (Table 1). Approximately two-thirds of these (14) were positive for Leptospira spp. plus 1 other organism; TGR, S. enterica serogroup Typhi, or Brucella spp. One patient was positive for 3 different organisms: by culture for both Brucella and Leptospira and by real-time PCR for Rickettsia felis (Table 1). This highly unusual case also represents the first reported case of R. felis in Egypt.
The remaining proportion of combined infection patients (n = 167, 11.0%) had only one positive single acute sera without a convalescent serum sample, positive culture, and/or showed high IgM ELISA antibody titers against multiple pathogens, although a single high antibody titer does not confirm that an agent is the cause of the acute illness. Most of the latter samples were positive for both leptospirosis and brucellosis, and a fewer number were positive for typhoid fever and leptospirosis or TGR or both. Many patients (n = 30) were probably coinfected with 3 or 4 pathogens; S. enterica serogroup Typhi, Leptospira, TGR, and/or Brucella spp. (Table 2).
Twice as many of the confirmed coinfected patients (13) were males with a median age of 25 years (range 12–50). The majority of the coinfected patients (53%) had daily animal contact, reported seeing rodents around the home (79%), consumed raw milk or milk products (64%), and had tap water in their homes (68%). The occupations of the patients or the heads of households were primarily day laborers (53%) and farmers (36%). Our study also found that exposure to rodents and cats was significantly associated with murine typhus positivity (P < 0.03 and P < 0.05, respectively), and rodents were also found to be significantly associated with Leptospira positivity (P < 0.01). Finally, the availability of municipal water was significantly (P < 0.01) associated with protection against leptospirosis.
DISCUSSION
Very little literature exists that describes possible concurrent AFI infections in this population. The results obtained in this study suggest that serial or concurrent infection with different pathogenic AFI agents is not uncommon in Egypt, possibly due to environmental and societal factors that favor the spread of these diseases. Taking into consideration the cultural practices of people in Egypt and the daily contact with animals, insects, and contaminated water sources, in addition to the consumption of unpasteurized milk products, several zoonotic diseases have the potential to cause AFI.1 A number of factors favor the spread of infectious diseases and facilitate the occurrence of simultaneous or multiple zoonoses. The majority of the coinfected patients in this study lived in high-density agricultural areas of Egypt and worked as day laborers and farmers. Farm animals are part of many households in the villages of rural Egypt and are used for work in the field and in the production of meat and milk. These animals are rarely vaccinated or treated with insecticides, and consumption of raw milk and cheese is common.1,14
Previous studies in Egypt have shown that rats and squirrel fleas, cats and their fleas, and other insect vectors have been involved in the transmission of R. typhi.15,16 Rats live around trash disposal sites, agricultural lands, and canals or streams dug off the Nile River for irrigation or drainage. Contact with such peridomestic animals, including rodents, is unequivocal in the transmission of diseases that cause febrile illness, including brucellosis, leptospirosis, and murine typhus. Also, because of the lack of public health awareness, water from unclean sources is frequently used for drinking, cooking, and washing, despite the presence of municipal water.
CONCLUSION
In this study, we determined the proportion of AFI due to concurrent infections using gold-standard laboratory tests. Detection of these coinfections at the time of presentation is often difficult, especially with limited laboratory support. Although identifying AFI coinfection is challenging, fortunately, therapy with doxycycline, an inexpensive but useful antibiotic, has been shown to be effective against leptospirosis, brucellosis, typhoid fever, and rickettsiosis.17 Therefore, clinicians should consider the use of doxycycline for AFI patients in Egypt who do not respond to other therapies. Analysis of cases from this project will continue so that a better picture of the prevalence of patients with concurrent or serial zoonotic disease exposure can be obtained. In addition, the collected data will be used to assist AFI prevention and control strategies currently used in Egypt.
Positive test results for acute febrile illness patients (n = 20) with confirmed coinfection with 2 or more organisms
| Coinfection | ||||
|---|---|---|---|---|
| No. of patients | Brucella | Leptospira | Rickettsia | Typhoid |
| * Refers to ≥ 4-fold change in acute and convalescent samples. | ||||
| 1 | Culture | Culture, PCR | PCR | |
| 2 | Culture, PCR | ELISA ≥ 4-fold change* | ||
| 2 | Culture | ELISA ≥ 4-fold change* | ||
| 2 | ELISA ≥ 4-fold change* | ELISA ≥ 4-fold change* | ||
| 2 | ELISA ≥ 4-fold change* | ELISA ≥ 4-fold change* | ||
| 1 | MAT ≥ 800-fold change* | ELISA ≥ 4-fold change* | ||
| 2 | Culture | ELISA ≥ 4-fold change* | ||
| 1 | Tube agglutination ≥ 4-fold change* | ELISA ≥ 4-fold change* | ||
| 2 | Culture | Culture, PCR | ||
| 1 | Tube agglutination ≥ 4-fold change* | Culture, PCR | ||
| 1 | ELISA ≥ 4-fold change* | Culture | ||
| 1 | ELISA ≥ 4-fold change* | ELISA ≥ 4-fold change* | ||
| 1 | Culture, PCR | Culture | ||
| 1 | ELISA > 4-fold change* | Culture | ||
Acute febrile illness patients (n = 167) with probable coinfection with 2 or more organisms
| No. of patients | Organism 1 | Organism 2 | Organism 3 | Organism 4 |
|---|---|---|---|---|
| * Tube agglutination, titer ≥ 320. | ||||
| † MAT, titer ≥ 800. | ||||
| ‡ Rickettsia ELISA, OD value > 0.2 or Typhoid ELISA, OD value > 0.3. | ||||
| 55 | Typhoid* | Leptospira† | ||
| 48 | Brucella* | Leptospira† | ||
| 23 | Typhoid* | Leptospira† | Rickettsia‡ | |
| 15 | Typhoid* | Brucella* | ||
| 9 | Brucella* | Rickettsia‡ | ||
| 5 | Typhoid* | Brucella* | Leptospira† | |
| 5 | Brucella* | Rickettsia‡ | Typhoid‡ | |
| 5 | Leptospira† | Rickettsia‡ | ||
| 2 | Typhoid* | Brucella* | Leptospira† | Rickettsia‡ |
Address correspondence to Tina M. Parker, United States Naval Medical Unit No. 3, PSC 452, Box 124, FPO AE, Cairo, Egypt 09835. E-mail: tina-parker@uiowa.edu
Authors’ addresses: Tina M. Parker, Ahmed Samir, Tharwat Ismail, Moustafa Abdel Fadeel, Momtaz O. Wasfy, and Guillermo Pimentel, U.S. Naval Medical Research Unit No. 3, PSC 452 BOX 5000 (Attn: Code 304A), FPO AE 09835, Telephone: +2-02-342-1375, Fax: +2-02-342-8933, E-mails: tina-parker@uiowa.edu or parker.dr@gmail.com, ahmed.samir@namru3.med.navy.mil, ismailt@namru3.med.navy.mil, fadeelm@namru3.med.navy.mil, wasfym@namru3.med.navy.mil, and pimentelg@namru3.med.navy.mil. Clinton K. Murray, Brooke Army Medical Center, 3851 Roger Brooke Drive, 7 East, Fort Sam Houston, TX 78234, Telephone: +1 (210) 916-3847, Fax: +1 (210) 916-0388, E-mail: Clinton.Murray@amedd.army.mil. Allen L. Richards and Ju Jiang, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, Telephone: (301) 319-7668, Fax: +1 (301) 319-9458, E-mails: richardsa@nmrc.navy.mil and jiangj@nmrc.navy.mil.
Copyright statement: Some of the authors are employees of the U.S. government, and this work was prepared as part of their official duties. Title 17 U.S.C. §105 provides that “Copyright protection under this title is not available for any work of the United States Government.” Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person’s official duties.
Acknowledgments: This work was supported by Work Unit Number (WUN) 847705-25GB-3906 (T.M.P., M.O.W., M.A.F., T.I., and G.P.) and DoD GEIS WUN 847705.82000.25GB.A0074 (J.J. and A.L.R.). We are grateful to Mohammad A. Maksoud for his laboratory assistance.
Disclaimer: The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the U.S. Navy Department or Egypt.
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