Figure 1

Figure 1. . . Oocysts (arrows), stained by using the Ziehl-Neelsen method, in sputum from the patient with HIV.

In September 2004, a 58-year-old man, who received an HIV 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 ≈10 mL of respiratory secretion was obtained. DNA was extracted as follows: 200 μL fluid was centrifuged and the pelleted material digested overnight at 65ºC with proteinase K in the presence of 10% sodium dodecyl sulfate. We then sequentially extracted the digested material with phenol, phenol-chloroform-isoamyl alcohol, and chloroform-isoamyl alcohol. The DNA were then precipitated with sodium acetate and ethanol, and after centrifugation, the pelleted DNA was dissolved in 50 µL of water.

Figure 2

Figure 2. . . Agarose gel electrophoresis of DNA fragments amplified with Cryptosporidium species–specific Lib13 primers (7). Ch, C. hominis; Cp, C. parvum; M, DNA molecular marker (Bioline, Randolph, MA, USA;...

For molecular typing, we first used the species-specific oligonucleotide PCR assay Lib13, 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, Lib13SF02 (5′-TTTTTTCATTAGCTCGCTTC-3′), a fragment of ≈400 bp, was amplified specifically from C. hominis DNA with the anti-sense primers Lib13SRT-1 (5′-ATTTATTAATTTATCTCTTACTT-3′) and from C. parvum DNA with Lib13SRT-2 (5′-ATTTATTAATTTATCTCTTCG-3′) (Figure 2). Amplifications were carried out in a PCR mixture of 10 μL 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 95ºC for 5 min, then 40 cycles at 95ºC for 30 s, 52ºC for 30 s, and 68ºC for 30 s. The mixture was then cooled to 4ºC.

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′-GTTGATCCTGCCAGTAGTC-3′) and 18SR (5′-TAAGGTGCTGAAGGAGTAAGG-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 ≈5 μm 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 (≈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/Lib13SRT2) 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).

We also analyzed the 18S small subunit rRNA gene by amplifying and sequencing an 18S rRNA fragment from the sputum DNA. Amplification and sequencing was concomitantly performed with the C. hominis and C. parvum DNA. A polymorphic site exists in C. hominis as a stretch of 10 to 12 thymines (T_10–12), while in C. parvum, the sequence is TA:::TATATTTT (146). The 18S rRNA polymorphic sequence found in the sputum sample DNA (GenBank accession no. DQ286403) is that of C. hominis, with a stretch of 11 Ts (Table). Few other nucleotide polymorphisms were found between the sequences (results not shown), which reflect intraisolate variations (14). The results with Lib13 assay and the partial 18S rRNA sequence analysis, therefore, identify the species of Cryptosporidium infecting the respiratory tract of this patient as C. hominis.

Human cryptosporidiosis is better known as an intestinal disease both in immunocompetent and immunocompromised persons. Little information exists, however, on human pulmonary disease caused by Cryptosporidium spp., which reflects either the low prevalence of pulmonary cryptosporidiosis or the lack of testing in immunocompetent hosts. Further, if performed, current diagnostic tests may not be sensitive enough to detect the parasite.

In immunocompetent children with intestinal cryptosporidiosis, respiratory symptoms have been noted more frequently than expected (5). Studies are needed in immunocompetent persons, especially children <2 years of age, who belong to a group at high risk for intestinal, and by extension pulmonary, cryptosporidiosis.

More information about which species of this pathogen infect humans and the pathogenic patterns each species produces is needed. As we determined here, C. hominis have the capacity to adapt to different physiologic environments, such as intestinal and respiratory tract tissues. Our findings provide additional evidence supporting the role of this species of Cryptosporidium as a human pathogen and the need to evaluate the importance of pulmonary cryptosporidiosis as a disease in the immunocompromised host.