Olivia 2009
- 1. Am. J. Trop. Med. Hyg., 80(1), 2009, pp. 51–54 Copyright © 2009 by The American Society of Tropical Medicine and Hygiene Short Report: Two Different Chitinase Genotypes in a Patient with an Amebic Liver Abscess: A Case Report Olivia Valenzuela, Patricia Morán, Fernando Ramos, Jorge I. Cardoza, Gabriela García, Alicia Valadez, Liliana Rojas, Adriana Garibay, Enrique González, and Cecilia Ximénez* Departamento de Ciencias Químico Biológicas, Universidad de Sonora, México; Departmento de Medicina Experimental, Facultad de Medicina Universidad Nacional Autónoma de Mexico, México DF; Departamento de Medicina Interna, Hospital General del Estado, Hermosillo Sonora, México Abstract. The present work deals with the identification of a patient with two liver abscesses containing two different strains of Entamoeba histolytica, as defined by chitinase gene plymorphisms. INTRODUCTION before hospital admission, the patient had been complain- ing of right upper quadrant abdominal pain, fever (> 38°C) Entamoeba histolytica is an important cause of morbidity and an unexplained weight loss of 10 kg (22 lbs). The clini- and mortality in Latin America, Asia, and Africa. The three cal diagnosis was ALA as confirmed by x-rays, a computed major clinical forms of E. histolytica infection are asymptom- tomography (CT) scan, and enzyme-linked immunosorbent atic colonization of the intestinal mucosa (asymptomatic cyst assay (ELISA), which indicated high levels (OD, 1.803) of passers), amebic colitis or dysentery in which the trophozo- serum anti-E. histolytica antibodies (IgG).15 An electrocar- ites invade the intestinal mucosa, and amebic liver abscesses diogram showed no abnormalities and the thoracic x-ray (ALAs) where trophozoites invade the intestinal mucosa and showed a slight elevation of the right hemi-diaphragm reach the hepatic parenchyma through the portal circulation (Figure 1A). The abdominal ultrasonography performed dur- system. ing admission showed a 13.1 cm long, 14.6 cm trans, and 9.3- In Mexico, ALA is associated with significant morbid- cm AP hypo-echoic area in the left hepatic lobe, which was ity and low but significant mortality.1 Although ALA grows indicative of a liver abscess. The right hepatic lobe showed inexorably in humans and is always fatal without appropriate only a slight increase in size (data not shown). treatment, current anti-amebic therapy2 can treat even large To improve the view of the right hepatic lobule, an abdom- amebic abscesses. inal CT scan was performed on day eight after admission. The high rate of intestinal re-infection in highly endemic A large left lobe abscess was clearly observed, and a sec- areas is mainly the result of repetitive exposure to Entamoeba ond independent abscess was detected in the right lobule parasites.3–8 The development of repeated ALA is more fre- (Figure 1B). Anti-amebic treatment with intravenous (IV) quent than previously thought.9–11 Furthermore, it has been metronidazole was established (500 mg every 8 hours) and reported that roughly 50% of ALA cases develop more than “intravenously” cefotaxime (1 g every 8 hours) was given for one ALA at the same time.12,13 antimicrobial coverage. Cultures of the aspirated material In the present work, we report an adult patient who devel- yielded negative results. oped two simultaneous and independent ALAs. The size of The clinical course after a 72-hour treatment was poor both abscesses necessitated drainage. The drained material because of the persistence of fever (up to 38°C) and abdom- was submitted to DNA extraction and subsequent polymerase inal pain. The physician in charge decided to drain both chain reaction (PCR) amplification of polymorphic regions in abscesses because of the risk of rupture. The procedure was the chitinase gene of E. histolytica species (Ehcht). The Ehcht guided by ultrasound. A multi-purpose catheter was placed in gene encodes a protein with a repeating structure at the amino each abscess. terminus.14 This degenerate heptapeptide repeat region exhib- Both catheters were removed after 15 days because of sig- its a polymorphism among isolates of Entamoeba species nificant clinical improvement and the patient was discharged with regard to the number and type of repeat copies. Results 3 days later. The patient remained under outpatient manage- of the polymorphism study of the Ehcht gene from both of ment with an oral regimen of metronidazole (500 mg every the patient’s aspirated ALAs suggest that the two indepen- 8 hours) and ciprofloxacine (500 mg every 12 hours) for dent liver abscesses were caused by two genetic variants of another 5 days. E. histolytica. The ELISA method used to detect and quantify E. his- tolytica antibody was previously reported.16 The cut-off point CASE REPORT for IgG (OD of 0.525) was defined as the best optical den- sity value obtained from the receiver operating characteristic The patient was a 48-year-old male with a history of dia- (ROC) curve for serum samples and for detection of anti- betes, alcoholism, and smoking for the last 20 years. The amebic antibodies.16 patient was admitted to the Hospital General del Estado The DNA was extracted from independent aspirate sam- in Hermosillo, Sonora, on September 28, 2007. For 10 days ples of each ALA (right and left hepatic lobules) using the QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA) in accor- dance with the provider’s protocol. * Address correspondence to Cecilia Ximénez, Departamento de Medicina Experimental, Facultad de Medicina, UNAM, Dr. Balmis Amplification of the highly polymorphic region of the 148, Col. Doctores, CP 06726, México D.F., México. E-mail: chitinase gene was done by PCR using primers described in cximenez@servidor.unam.mx Table 1. The PCR conditions were previously described.17 51
- 2. 52 VALENZUELA AND OTHERS Figure 1. A, Thoracic x-ray taken during patient admission. The image shows elevation of the hemidiaphragm. B, Computed tomography (CT) scan showing an image of both large abscesses in the left and right hepatic lobules. After electrophoresis, the gels were photographed for our E. histolytica HM1:IMSS is shown in Figure 2A. Sequences of records. The PCR products were purified using the GFX kit both left and right lobules abscesses are shorter than the E. (Amersham Pharmacia Biotech, Sao Paulo, Brazil) and then histolytica HM1:IMSS strain, there is also a nucleotide change sequenced at the University of Arizona (Genomic Analysis & (G by A) in position 109 on the HM1:IMSS strain (Figure 2B). Technology Core, Tucson, AZ). The sequences were aligned The sequence of Ehcht gene in the left hepatic lobule abscess with the reported sequences of E. histolytica present in the is identical to genotype H reported previously in ALA cases Gen Bank data base (Figure 2A and B). in Mexico (Gen Bank accession no. EF-445962). However, the Ehcht gene sequence obtained in the right hepatic lobule abscess is identical to the one on NIH-200 and K1 E. histolyt- RESULTS AND DISCUSSION ica strains reported by Ghosh and others.4 In the present work, we report a patient with ALA who Translation of the nucleotide sequences into amino acid developed two independent abscesses located in the right and sequences and the corresponding alignment is shown in left hepatic lobules (Figure 1A and B). The clinical findings Figure 2B and correlates with the nucleotide sequences men- were coincident with the presence of high levels of circulating tioned earlier. anti-amebic antibodies, as detected by ELISA. Together, these Even though simultaneous intestinal infections by E. his- results point to an amebic etiology for the liver abscess (OD tolytica and E. dispar have been previously reported,17,19–21 this at 490 nm of 1.8).15,16 was the first report that showed amebic infection by more than Entamoeba histolytica was confirmed as the causative agent one genetic variant of the E. histolytica species in the same by PCR analyses of the drainage material from both abscesses. patient. This phenomenon has been described for other par- Samples from the two abscesses contained fragments of the asitic infections, such as trypanosomiasis, leishmaniasis, and Ehcht gene, the amplified region was located between the sig- malaria.22,23 nal sequence and the catalytic site of the protein, which has It is known that ALA occurs without any colitis symptoms heptapeptide repetitive sequences rich in hydrophilic amino and without any detectable parasites in the colon. On the acids (Ser, Glu, Ssp, Hys, and His). However, some hydro- basis of these clinical observations, Charmot24 emphasized phobic amino acids (Pro, Val, and Ile) were also present in the possibility that E. histolytica hepatotropic strains may the sequences.18 The alignment of the respective nucleotide exist by mentioning that “the localization, heterogeneity, and sequences of the PCR products obtained from material of the experimental data associated with clinical expression sug- both left and right hepatic lobules and the reported sequences gest the existence inside the species E. histolytica of various in the Gen Bank database (accession no. XM 647113.1) for strains: non-pathogenic one, invasive for the intestine mucous membrane ones, and invasive for liver tissue ones.” Recently, Ali and others25 genotyped E. histolytica strains from fecal and Table 1 aspirated ALA samples of the same patient and demonstrated Primer sequences the existence of co-infection by different types of strains. Their Primer Sequence results, together with our finding, have opened a series of pos- CHI5 GAAS*AACAGAAGGAACACCAGG sible explanations for this phenomenon. Ali’s group used poly- CHI-Eh/Ed3 TCTGTATTGTGCCCAATT morphic intergenic regions (short tandem repeats) associated Hsp 1 GAGTTCTCTTTTTATACTTTTATATGTT with tRNA genes as the target for genotyping the strains. The Hsp 2 ATTAACAATAAAGAGGGAGGT authors mentioned that their results were compatible with a StgaD-H5 AAATCCTGCCACTGTCGTAA StgaD-H3 AATCCCCGTTGAAGAGTTCT possible new infection during the lapse between the first inoc- SQ 5 GTGGTCTAAGGCGTGTGACT culum and the development of ALA symptoms, an account that SQ H3 GTGGGACCACTTTTTATACCTA is reasonable in endemic regions. Another possible explanation *S=C+G is the existence of ecologic niches in endemic environments in
- 3. TWO DIFFERENT EhCHT GENOTYPES IN AN AMEBIC LIVER ABSCESS 53 Figure 2. A, Nucleotide sequence alignment of the polymerase chain reaction (PCR) amplification products amplified by CHI primers. The DNA was independently extracted from the drainage material of the left and right hepatic lobule abscesses. B, Alignment of the deduced amino acid sequences obtained from the PCR products of the Eh chitinase gene. The DNA from the E. histolytica HM1:IMSS strain was simultaneously studied as a reference strain. The corresponding heptapeptides were marked in color. This figure appears in color at www.ajtmh.org. which genetically heterogeneous Entamoeba species could Medicina Interna, Hospital General del Estado, Hermosillo Sonora, exist (i.e., E. histolytica and E. dispar species, as was previously México, Boulevard Luis Encinas y Reyes Col. Centro. CP 83000, Tel/Fax: +52 662 2121422. mentioned, or intraspecies genetic variants).6,19–21 Another explanation mentioned by Ali and others is that a Reprint requests: Cecilia Ximénez, Departamento de Medicina recombination event may have occurred on the parasite DNA Experimental, Facultad de Medicina, UNAM, Dr. Balmis 148, Col. Doctores, CP 06726, México D.F., México, Tel: +5255-56-23-26-66, during the migration process from the intestine to the liver.25 Fax: +5255-56-23-26-79, E-mail: cximenez@servidor.unam.mx. These events are relatively frequent in multiple copy genes, such as the STR sequences in tRNA genes.26,27 These types of REFERENCES chromosomal structures are less stable than the sequences located in single copy genes, such as the chitinase gene. 1. Mexican Health Minister. Available at: http://dgepi.salud.gob. In fact, we tested three loci of the intergenic region associ- mx.anuario/index.html. 2. Stanley SL Jr, 2003. Amoebiasis. 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