Cryptosporidium hominis infection of the human respiratory tract.Cryptosporidium cryptosporidium (krĭp'tōspərĭd`ēəm), genus of protozoans having at least four species; they are waterborne parasites that cause the disease cryptosporidiosis. oocysts, observed in a natural sputum sputum /spu·tum/ (spu´tum) [L.] expectoration; matter ejected from the trachea, bronchi, and lungs through the mouth.
sputum cruen´tum bloody sputum. sample of a patient with HIV HIV (Human Immunodeficiency Virus), either of two closely related retroviruses that invade T-helper lymphocytes and are responsible for AIDS. There are two types of HIV: HIV-1 and HIV-2. HIV-1 is responsible for the vast majority of AIDS in the United States. , were further studied by using DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. markers to determine the species of the parasite. C. hominis was identified as the species infecting the patient's respiratory tract respiratory tract
The air passages from the nose to the pulmonary alveoli, including the pharynx, larynx, trachea, and bronchi.
Respiratory tract , a finding that strengthens evidence regarding this pathogen's role in human disease.
Intestinal cryptosporidiosis Cryptosporidiosis Definition
Cryptosporidiosis refers to infection by the sporeforming protozoan known as Cryptosporidia. Protozoa are a group of parasites that infect the human intestine, and include the better known Giardia. is a common parasitic disease that causes self-limiting diarrhea in immunocompetent im·mu·no·com·pe·tent
Having the normal bodily capacity to develop an immune response following exposure to an antigen.
im persons (1). Higher frequencies of Cryptosporidium infection are observed in immunocompromised immunocompromised /im·mu·no·com·pro·mised/ (-kom´pro-mizd) having the immune response attenuated by administration of immunosuppressive drugs, by irradiation, by malnutrition, or by certain disease processes (e.g., cancer). humans, and the main clinical pattern of the infection in these persons is a chronic, life-threatening secretory secretory /se·cre·to·ry/ (se-kre´tah-re) (se´kre-tor?e) pertaining to secretion or affecting the secretions.
Relating to or performing secretion. 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 Noun 1. respiratory tract infection - any infection of the respiratory tract
infection - the pathological state resulting from the invasion of the body by pathogenic microorganisms 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.
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 aliquot (al-ee-kwoh) adj. a definite fractional share, usually applied when dividing and distributing a dead person's estate or trust assets. (See: share) 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 proteinase /pro·tein·ase/ (pro´ten-as?) endopeptidase.
A protease that begins the hydrolytic breakdown of proteins usually by splitting them into polypeptide chains. K in the presence of 10% sodium dodecyl sulfate Sodium dodecyl sulfate (or sulphate) (SDS or NaDS) (C12H25NaO4S),is an anionic surfactant that is used in household products such as toothpastes, shampoos, shaving foams and bubble baths for its thickening effect and its ability to . We then sequentially extracted the digested material with phenol phenol (fē`nōl), C6H5OH, a colorless, crystalline solid that melts at about 41°C;, boils at 182°C;, and is soluble in ethanol and ether and somewhat soluble in water. , phenolchloroform-isoamyl alcohol, and chloroform-isoamyl alcohol. The DNA was then precipitated with sodium acetate and ethanol, and after centrifugation Centrifugation
A mechanical method of separating immiscible liquids or solids from liquids by the application of centrifugal force. This force can be very great, and separations which proceed slowly by gravity can be speeded up enormously in centrifugal , 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 PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (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 Abi (ā`bī) [short for Abijah], in the Bible, King Hezekiah's mother.
(Application Binary Interface) A specification for a specific hardware platform combined with the operating system. , Foster City, CA, USA) with initial denaturation denaturation, term used to describe the loss of native, higher-order structure of protein molecules in solution. Most globular proteins exhibit complicated three-dimensional folding described as secondary, tertiary, and quarternary structures. 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 TOPO Tri-N-Octylphosphine Oxide
TOPO Trioctyl-Phosphine Oxide
ToPo Torposten (German Military Gate Post)
TOPO Tunable Optical Parametric Oscillator 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 Formed by a merger between the Richmond Professional Institute and the Medical College of Virginia in 1968, VCU has a medical school that is home to the nation's oldest organ transplant program. . All DNA sequences were analyzed by using Sequencher (Gene Codes Co., Ann Arbor, MI, USA).
Figure 1 shows fuchsia fuchsia: see evening primrose.
Any of about 100 species of flowering shrubs and trees in the genus Fuchsia (family Onagraceae), native to tropical and subtropical regions of Central and South America and to New Zealand and Tahiti. (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 cal·i·brate
tr.v. cal·i·brat·ed, cal·i·brat·ing, cal·i·brates
1. To check, adjust, or determine by comparison with a standard (the graduations of a quantitative measuring instrument): 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 (LIBF LIBF London International Book Fair (UK)
LIBF Lust-Induced Brain Freeze (Dave Barry) 02/Lib13SRT (1) (Source Routing Transparent) An IEEE-standard that provides bridging between Ethernet and Token Ring networks. Ethernet LANs use transparent bridging, and Token Ring LANs use source route bridging (SRB). 1), 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).
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.sub.10-12]), while in C. parvum, the sequence is TA:::TATATTTT (14). 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 Libl3 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.
We thank Giovanni Widmer and Sultan Tanriverdi for the C. parvum DNA and helpful discussions and Carmen Carmen
throws over lover for another. [Fr. Lit.: Carmen; Fr. Opera: Bizet, Carmen, Westerman, 189–190]
See : Faithlessness
the cards repeatedly spell her death. [Fr. Chambel, Claudio Rojas, and Sergio Guillermo Tula for technical assistance.
This work was supported by the Cryptosporidium Project 2004 for the Unidad Docente de Parasitologia, Faculty of Medicine, Universidad de Chile, and by funds from the National Institutes of Health (grant no. U01 AI46418).
Dr Mercado is assistant professor at the Unidad Docente de Parasitologia, Facultad de Medicina, Universidad de Chile, Santiago. His research interests focus on human parasites and the epidemiology of infectious diseases.
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Relating to, resembling, or characteristic of deer.
[Latin cerv origin for healthy adults and interferon-[gamma] knockout mice. J Infect Dis. 2002;185:1320-5.
(14.) Gibbons-Matthews C, Prescott AM. Intra-isolate variation of Cryptosporidium parvum small subunit ribosomal RNA genes from human hosts in England. Parasitol Res. 2003;90:439-44.
Address for correspondence: Ruben Mercado, Unidad Docente de Parasitologia, Facultad de Medicina, Universidad de Chile, PO Box 33052, Correo 33, Santiago, Chile; email: email@example.com
Ruben Mercado, * Gregory A. Buck, ([dagger]) Patricio A. Manque man·qué
Unfulfilled or frustrated in the realization of one's ambitions or capabilities: an artist manqué; a writer manqué. , ([dagger]) and Luiz Shozo Ozaki ([dagger])
* Facultad de Medicina, Universidad de Chile, Santiago, Chile; and ([dagger]) Virginia Commonwealth University, Richmond, Virginia, USA
Table. Nucleotide sequences at the T polymorphic site of the 18S rRNA of the pulmonary sample of Cryptosporidium (Chile01), C. parvum (Cp), and C. hominis (Ch) Isolate 18S rRNA sequence at 670-710 Chile01 5'-CATAATTCATATTACTATTTTTTTTTTTA GTATATGAAATT-3' Cp 5'-CATAATTCATATTACTA:::TATATTTTA GTATATGAAATT-3' Ch 5'-CATAATTCATATTACTA:TTTTTTTTTTA GTATATGAAATT-3'