Cryptosporidium hominis infection of the human respiratory tract

Emerging Infectious Diseases, March, 2007 by Ruben Mercado, Gregory A. Buck, Patricio A. Manque, Luiz Shozo Ozaki

Cryptosporidium oocysts, observed in a natural sputum sample of a patient with HIV, were further studied by using DNA markers to determine the species of the parasite. C. hominis was identified as the species infecting the patient's respiratory tract, a finding that strengthens evidence regarding this pathogen's role in human disease.

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Intestinal cryptosporidiosis is a common parasitic disease that causes self-limiting diarrhea in immunocompetent persons (1). Higher frequencies of Cryptosporidium infection are observed in immunocompromised humans, and the main clinical pattern of the infection in these persons is a chronic, life-threatening secretory diarrhea (2).

At least 8 species of Cryptosporidium are described as infecting humans. C. hominis and C. parvum are the most frequently observed in intestinal infections in humans (3). C. meleagridis is also detected both in immunocompetent and immunodeficient patients, although at a lower rate than C. parvum (4).

Respiratory tract infection by Cryptosporidium spp. has been described for immunodeficient persons, most all of whom were coinfected with HIV. However, pulmonary cryptosporidiosis was also described in patients without HIV infection (5, 6). In all cases, no systematic identification of the species of Cryptosporidium was pursued except by Meamar et al. (7), in which the parasite was identified as C. parvum.

We describe the detection and identification of C. hominis in the respiratory secretions of a patient with HIV (sample Chile01). We used an oligonucleotide species-specific method and sequencing of parts of the 18S rRNA gene to determine the species of Cryptosporidium. Both analyses showed that the species of Cryptosporidium present in the pulmonary secretion of this patient was C. hominis.

The Study

In September 2004, a 58-year-old man, who received an HIV-positive diagnosis in 1996, was hospitalized with respiratory symptoms characterized by persistent cough. Cryptosporidium oocysts were detected in a sputum sample from the patient by using Ziehl-Neelsen stain (Figure 1). An aliquot of [approximately equal to] 10 mL of respiratory secretion was obtained. DNA was extracted as follows: 200 [micro]L fluid was centrifuged and the pelleted material digested overnight at 65 [degrees] C with proteinase K in the presence of 10% sodium dodecyl sulfate. We then sequentially extracted the digested material with phenol, phenolchloroform-isoamyl alcohol, and chloroform-isoamyl alcohol. The DNA was then precipitated with sodium acetate and ethanol, and after centrifugation, the pelleted DNA was dissolved in 50 [micro]L of water.

[FIGURE 1 OMITTED]

For molecular typing, we first used the species-specific oligonucleotide PCR assay Lib l3, as described by Tanriverdi et al. (8), with a new sense oligonucleotide primer based on the genome sequences of C. hominis (9) and C. parvum (10). The new primer, Libl3SF02 (5'-TTTTTTCATTAGCTCGCTTC-3'), a fragment of [approximately equal to] 400 bp, was amplified specifically from C. hominis DNA with the anti-sense primers Lib 13 SRT-1 (5'-ATTTATTAATTTA TCTCTTACTT-3') and from C. parvum DNA with Lib13 SRT-2 (5'-ATTTATTAATTTATCTCTTCG-3') (Figure 2). Amplifications were carried out in a PCR mixture of 10 [micro]L containing 0.25 mmol/L of each dNTP, 300 pmol/L of each olignucleotide, and 1 unit of Taq DNA polymerase (HotMaster, Eppendorf, New York, NY, USA). Temperature cycling was performed on a GeneAmp PCR System (ABI, Foster City, CA, USA) with initial denaturation performed at 95[degrees]C for 5 min, then 40 cycles at 95 [degrees]C for 30 s, 52[degrees]C for 30 s, and 68[degrees]C for 30 s. The mixture was then cooled to 4 [degrees] C.

[FIGURE 2 OMITTED]

The region from bases 7 to 1036 (numbering according to C. hominis sequence GenBank no. L16996) of the 18S rRNA gene was sequenced from DNA fragments amplified using the primers 18SF (5'-GTTGATCCT GCCAGTAGTC-3') and 18SR (5'-TAAGGTGCTGAAG GAGTAAGG-3') and cloned into the TOPO TA vector (Invitrogen, Brandford, CT, USA) by using standard techniques. Automated sequencing was performed directly on the amplified fragments or on cloned fragments at the Nucleic Acid Research Facilities of Virginia Commonwealth University. All DNA sequences were analyzed by using Sequencher (Gene Codes Co., Ann Arbor, MI, USA).

Figure 1 shows fuchsia (Ziehl-Neelsen)-stained sputum from the patient. Three Cryptosporidium oocysts (arrows) with typical sizes [approximately equal to] 5 [micro]m in diameter are visible. Measurements were performed in a calibrated microscope as described by Mercado and Santander (11).

A Lib13 PCR assay (8) was performed on the DNA purified from the respiratory secretion material, and the results are shown in Figure 2. With the C. hominis specific-primer pair (LIBF02/Lib13SRT1), a fragment of the expected size ([approximately equal to] 400 bp) was amplified with the Chile01 isolate DNA (Figure 2, lane 2). With this primer pair, we also obtained amplification with the C. hominis isolate TU502 (12) DNA (Figure 2, lane 3) but not with the C. parvum isolate Moredun (13) DNA (Figure 2, lane 4). Conversely, the C. parvum-specific primer pair (LIBF02/Libl3SRT2) amplified a fragment only with the C. parvum DNA (Figure 2, lane 8). No amplification was observed with the Chile01 (Figure 2, lane 6) or the C. hominis DNA (Figure 2, lane 7). DNA sequencing of the amplified fragments confirmed the polymorphism to be that of C. hominis (results not shown).