Parasitology Research

, Volume 98, Issue 6, pp 593–595

Cryptosporidium parvum bovine genotype oocysts in the respiratory samples of an AIDS patient: efficacy of treatment with a combination of azithromycin and paromomycin

Authors

  • Ahmad-Reza Meamar
    • Department of Medical Parasitology and Mycology, Division of Intestinal and Genital Protozoal Diseases, School of Public Health and Institute of Public Health ResearchTehran University of Medical Sciences
  • Mostafa Rezaian
    • Department of Medical Parasitology and Mycology, Division of Intestinal and Genital Protozoal Diseases, School of Public Health and Institute of Public Health ResearchTehran University of Medical Sciences
  • Sasan Rezaie
    • Department of Medical Parasitology and Mycology, Division of Intestinal and Genital Protozoal Diseases, School of Public Health and Institute of Public Health ResearchTehran University of Medical Sciences
  • Minoo Mohraz
    • Department of Infectious Diseases, Imam Khomeini HospitalTehran University of Medical Sciences
  • Eshrat B. Kia
    • Department of Medical Parasitology and Mycology, Division of Intestinal and Genital Protozoal Diseases, School of Public Health and Institute of Public Health ResearchTehran University of Medical Sciences
  • Eric R. Houpt
    • Division of Infectious Diseases and International HealthUniversity of Virginia Health System
    • Department of Medical Parasitology and Mycology, Division of Intestinal and Genital Protozoal Diseases, School of Public Health and Institute of Public Health ResearchTehran University of Medical Sciences
    • Division of Infectious Diseases and International HealthUniversity of Virginia Health System
Short Communication

DOI: 10.1007/s00436-005-0097-4

Cite this article as:
Meamar, A., Rezaian, M., Rezaie, S. et al. Parasitol Res (2006) 98: 593. doi:10.1007/s00436-005-0097-4

Abstract

Cryptosporidium has been recognized as an emerging zoonotic agent of intestinal cryptosporidiosis leading to diarrhea, malabsorption syndrome, and weight loss in AIDS patients. In the present case, oocysts of zoonotic Cryptosporidium parvum were detected in the sputum and stool samples of an AIDS patient with a 3-month history of intestinal cryptosporidiosis. The oocysts were detected by modified Ziehl–Neelsen staining; confirmation was achieved by nested polymerase chain reaction (PCR), targeting the most polymorphic region of the 18S rRNA gene. Genotyping was done by restriction endonuclease digestion of the PCR product. The zoonotic C. parvum bovine genotype was identified in both intestinal and respiratory samples. Treatment with both azithromycin and paromomycin resulted in improvement of both intestinal and respiratory symptoms, as well as the elimination of the parasite. This is the first report of the identification of Cryptosporidium sp. oocysts in the respiratory samples obtained from an AIDS patient in Iran. Pulmonary cryptosporidiosis should be considered whenever an AIDS patient with intestinal cryptosporidiosis develops respiratory symptoms.

Introduction

The genus Cryptosporidium includes zoonotic apicomplexan protozoan parasites, which infects the gastrointestinal and respiratory tracts of a wide variety of animals and humans (Fayer and Ungar 1986; Chen et al. 2002). Since the first report of human cryptosporidiosis infection in 1976, 15 valid species have been recognized, including Cryptosporidium hominis, Cryptosporidium muris, Cryptosporidium andersoni, Cryptosporidium parvum, Cryptosporidium wrairi, Cryptosporidium felis, Cryptosporidium bovis, Cryptosporidium suis, and Cryptosporidium canis (in mammals); Cryptosporidium baileyi, Cryptosporidium meleagridis, and Cryptosporidium galli (in birds); Cryptosporidium serpentis and Cryptosporidium saurophilum (in reptiles); and Cryptosporidium molnari (in fish) (Clark 1999; Hunter and Nichols 2002). Although it appears that some strains of the parasite adapted to certain hosts, cross-strain infectivity also occurs and may or may not be associated with illness. The main causative agents of human cryptosporidiosis are C. hominis and C. parvum. The old designations for C. parvum are genotypes 1 and 2, which are now C. hominis and C. parvum, respectively. Infections of immunocompetent patients are usually associated with C. hominis; infections of immunodeficient [human immunodeficiency virus (HIV)-positive] patients can also be correlated with various animal genotypes or even primarily animal pathogenic species of Cryptosporidium (e.g., C. meleagridis, C. canis, and C. felis) (Fayer et al. 2000).

While the small intestine is the site most commonly affected, symptomatic Cryptosporidium infections have also been found in other organs, including other digestive tract organs, the lungs, and, infrequently, conjunctiva. Compared to digestive disorders, respiratory disease linked to Cryptosporidium has not been very well documented in patients with HIV/AIDS. The species or strains infecting the respiratory system are not currently distinguished from those infecting the intestines (Dupont et al. 1996).The human disease is usually self-limiting in immunocompetent persons but may cause severe and chronic life-threatening gastroenteritis in immunosuppressed patients, including HIV/AIDS patients. The advent of the AIDS epidemic and the recognition of waterborne outbreaks of cryptosporidiosis led to the inclusion of Cryptosporidium as an emerging infectious pathogen.

Pulmonary or tracheal cryptosporidiosis is characterized by coughing, often accompanied by a low-grade fever and severe intestinal symptoms (Fayer and Ungar 1986). In immunodeficient individuals, especially in AIDS patients, the disease may remain for life, with severe watery diarrhea contributing to death (Brady et al. 1984). The invasion of the pulmonary system by parasites may also be fatal. Here, we report the detection of C. parvum oocysts in the sputum and stool samples of an AIDS patient with respiratory symptoms. So far, a limited number of isolates have been typed from developing countries and especially from HIV-infected people (Morgan et al. 2000; Peng et al. 2001), yet cryptosporidiosis is endemic in most tropical regions. Extensive genotyping of isolates from different parts of the world is therefore crucial for a more precise mapping of the epidemiology of Cryptosporidium. The present study has targeted the most polymorphic region of the 18S rRNA gene in Cryptosporidium for precise parasite identification. This is the first documented case of pulmonary cryptosporidiosis in Iran.

In December 2004, a 37-year-old man, living in Ilam province of southwestern Iran, was admitted to Tehran Imam Khomeini Hospital. He had a 3-month history of fever, productive cough, and diarrhea, with 20 bowel movements per day, and weight loss of 17 kg during the previous 3 months. He also had cachexia and oral candidiasis. After serological examination, anti-HIV antibodies were detected; CD4 cells numbered 120 per cubic millimeter, and the tuberculin test was found to be negative. Giemsa staining of a sputum sample for Pneumocystis carinii was negative. No other parasites or pathogenic bacteria were found by either stool examination or culture. However, the Ziehl–Neelsen acid-fast staining method revealed the presence of Cryptosporidium sp. oocysts in both stool and sputum samples. Chest radiography failed to detect the presence of infection.

A diarrheic stool sample and an induced sputum sample were obtained at the time of hospitalization. The stool sample was fixed in 10% formalin and processed using the formalin–ether concentration technique. In addition, a second smear was made, air-dried, and stained using a modified acid-fast procedure to detect Cryptosporidium and other coccidian protozoa under a light microscope (×1,000). A few grams of the stool sample were frozen for subsequent polymerase chain reaction (PCR) analysis. Additionally, sputum was treated with 0.1 N KOH for 20 min and washed three times with phosphate-buffered saline. Thin smears were prepared on glass microscope slides using 20 μl of sediment. The smears were stained by using Giemsa and Ziehl–Neelsen staining methods.

Stool DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) spin column method following the manufacturer’s instructions. Cryptosporidium species and the C. parvum genotype were determined by nested PCR of a SSU-rRNA gene fragment and restriction fragment length polymorphism (RFLP) analysis with the endonucleases VspI and SspI as described elsewhere (Xiao et al. 1999). Primers, amplifying the 1,325-bp fragment of SSU-rRNA gene, were used for the initial PCR, followed by nested PCR performed using a second set of primers located in an initially amplified fragment. Fifteen microliters of the secondary PCR products were digested with both SspI and VspI (Promega, USA) restriction enzymes in a final volume of 20 μl for 2 h at 37°C. Digested products (10 μl) were fractioned and visualized on 2% agarose (Merck, Dramstad, Germany) gel containing 0.2 μg/ml ethidium bromide (Fig. 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs00436-005-0097-4/MediaObjects/436_2005_97_Fig1_HTML.gif
Fig. 1

Genotyping of the C. parvum parasite by nested PCR-RFLP procedure based on the SSU-rRNA gene digested with SspI and VspI. Lane 3, molecular marker V (Boehringer, Mannheim). Lanes 1 and 2, C. parvum bovine genotype (positive control) digested with VspI (lane 2) and SspI (lane 1). Lanes 4–6, C. parvum bovine genotype from patient stool (lane 4) and sputum (lane 5), digested with Vspl (lanes 5 and 6) and Sspl (lane 4). A 2% agarose gel stained with ethidium bromide and visualized by UV transilluminator

Cryptosporidium spp. is being increasingly recognized as a cause of disease among AIDS patients. Extraintestinal cryptosporidiosis, especially of the biliary and respiratory tract, is likely in the course of an intestinal involvement, whereas it is rare without such localization. Although the pathogenesis of cryptosporidial lung infection is still unclear, it was recently shown that Cryptosporidium organisms could develop in the bronchial epithelium of calves (Mascaro et al. 1994). Although intestinal Cryptosporidium sp. organisms are not usually invasive, oocysts have been found inside macrophages, which can have defective phagocyte-killing ability. As a matter of fact, Cryptosporidium organisms can multiply in macrophages in vitro (Martinez et al. 1992), suggesting that extraintestinal parasites might spread via circulating phagocytes.

Parasitic infections, including enteric cryptosporidiosis, microsporidiosis and cyclosporiasis, and Strongyloides stercoralis hyperinfection syndrome have already been reported from AIDS patients in Iran (Zali et al. 2004). However, this report is the first documented case of pulmonary cryptosporidiosis associated with enteric symptoms in an AIDS patient in Iran. As previously shown, it is possible that radiography failed to detect pulmonary cryptosporidiosis (Clavel et al. 1996), as in the case of a patient whose chest radiography remained normal throughout the clinical course of the disease.

After many years of research on strategies and drugs for treatment and control of cryptosporidiosis, there are no consistently effective, approved products for either animals or humans, although in recent years, nitazoxanide has been approved in the USA for treatment of cryptosporidiosis in children. Some drugs have proven toxic at doses required to reduce parasite multiplication, others have shown some efficacy only in animal models, and most others have shown no efficacy (Fayer 2004). However, paromomycin appears to have modest activity against C. parvum based on experimental data and clinical experience (Griffith 1998). Although the effectiveness of the combination of paromomycin and azithromycin in the treatment of cryptosporidiosis has not been definitively established (White et al. 1994), we selected an empiric trial of the combination of these two antimicrobial agents in the treatment of this patient. On hospital day 5, chemotherapy with azithromycin (500 mg twice a day, given orally) and paromomycin (500 mg twice a day, given orally) was started. Consequent sputum and stool examination were found to be negative for Cryptosporidium oocysts on hospital day 32. After a clear clinical improvement (disappearance of fever, respiratory symptoms, and diarrhea), the patient was discharged from the hospital, and the treatment was continued with azithromycin and paromomycin, as well as trimethoprim sulfamethoxazole prophylaxis for 1 month at the same dosage. This report serves to inform clinicians that different zoonotic species of Cryptosporidium can infect the lungs, producing cough and thick mucus, with or without radiographic findings. Also, we suggest that combined treatment with azithromycin and paromomycin should be further evaluated regarding its efficacy in reducing or eliminating AIDS-related cryptosporidial infection. To conclude, it is strongly recommended that whenever a patient with intestinal cryptosporidiosis develops respiratory symptoms, pulmonary cryptosporidiosis should be included in the differential diagnosis.

The study was conducted at the Department of Medical Parasitology and Mycology, Division of Molecular Biology, School of Public Health and Institute of Public Health Research, Tehran University of Medical Sciences, as part of the Ph.D. dissertation of A.R.M. We are grateful to Ronald Fayer, USDA, for critical reading and helpful discussion.

Copyright information

© Springer-Verlag 2006