Absence of indoleamine 2,3­dioxygenase 2 promotes liver regeneration after partial hepatectomy in mice.

The partial loss of liver due to liver transplantation or acute liver failure induces rapid liver regeneration. Recently, we reported that the selective inhibition of indoleamine 2,3­dioxygenase (Ido) 1 promotes early liver regeneration. However, the role of Ido2 in liver regeneration remains unclear. Wild­type (WT) and Ido2­deficient (Ido2­KO) mice were subjected to 70% partial hepatectomy (PHx). Hepatocyte growth was measured using immunostaining. The mRNA expression of inflammatory cytokines and production of kynurenine in intrahepatic mononuclear cells (MNCs) were analyzed using reverse transcription­quantitative PCR and high­performance liquid chromatography. The activation of NF­κB was determined by both immunocytochemistry and western blotting analysis. The ratio of liver to body weight and the frequency of proliferation cells after PHx were significantly higher in Ido2­KO mice compared with in WT mice. The expression of IL­6 and TNF­α in MNCs were transiently increased in Ido2­KO mice. The nuclear transport of NF­κB was significantly higher in peritoneal macrophages of Ido2­KO mice compared with WT mice. These results suggested that Ido2 deficiency resulted in transiently increased production of inflammatory cytokines through the activation of NF­kB, thereby promoting liver regeneration. Therefore, the regulation of Ido2 expression in MNCs may play a therapeutic role in liver regeneration under injury and disease conditions.

[A Case of Neuroendocrine Carcinoma Originating from the Papilla of Vater].

A woman in her late 70s with fatigue, nausea, and epigastric discomfort was found to have a tumor at the papilla of Vater through endoscopy. We performed subtotal stomach-preserving pancreaticoduodenectomy with regional lymph node dissection. The immunohistological analysis showed positive staining for chromogranin A, synaptophysin, and CD56. The definitive diagnosis was neuroendocrine carcinoma of the papilla of Vater. Although the patient declined adjuvant chemotherapy, she had to start chemotherapy with carboplatin and etoposide because multiple liver metastases, lymph node metastasis, and peritoneal dissemination occurred 6 months after surgery. We performed 6 courses of chemotherapy. However, progressive disease(PD)was assessed, and she died of cancer 13 months after the surgery. The prognosis of the disease is poor when surgery alone is performed. Adjuvant chemotherapy, in addition to surgery, may be necessary.

3-Hydroxykynurenine Regulates Lipopolysaccharide-Stimulated IL-6 Production and Protects against Endotoxic Shock in Mice.

Despite advances in our understanding of endotoxic shock, novel therapeutic interventions that can reduce the burden of sepsis remain elusive. Current treatment options are limited, and it is only through refinements in the ways that we deliver supportive care that mortality has fallen over the years. In this study, the role of kynurenine 3-monooxygenase (KMO) in immune regulation was examined in LPS-induced endotoxemia using KMO-/- and KMO+/+ mice treated with the KMO inhibitor Ro61-8048. We showed that LPS-induced or cecal ligation and puncture-induced mortality and hepatic IL-6 production increased in the absence of KMO, possibly involving increased activating transcription factor 4 (ATF4) signaling in hepatic macrophages. Moreover, treatment of septic mice with 3-hydroxykynurenine reduced mortality rates and inflammatory responses regardless of the presence or absence of KMO. According to our results, the administration of 3-hydroxykynurenine as part of the treatment approach for sepsis or as an adjuvant therapy might reduce the overproduction of IL-6, which is responsible for severe endotoxemia, and ultimately improve the survival rates of patients with sepsis.

Evaluation of red blood cell parameters provided by the UF-5000 urine auto-analyzer in patients with glomerulonephritis.

OBJECTIVES: The microscopic examination of hematuria, a cardinal symptom of glomerulonephritis (GN), is time-consuming and labor-intensive. As an alternative, the fully automated urine particle analyzer UF-5000 can interpret the morphological information of the glomerular red blood cells (RBCs) using parameters such as UF-5000 small RBCs (UF-%sRBCs) and Lysed-RBCs. METHODS: Hematuria samples from 203 patients were analyzed using the UF-5000 and blood and urine chemistries to determine the cut-off values of RBC parameters for GN and non-glomerulonephritis (NGN) classification and confirm their sensitivity to the IgA nephropathy and non-IgA nephropathy groups. RESULTS: The UF-%sRBCs and Lysed-RBCs values differed significantly between the GN and NGN groups. The cut-off value of UF-%sRBCs was >56.8% (area under the curve, 0.649; sensitivity, 94.1%; specificity, 38.1%; positive predictive value, 68.3%; and negative predictive value, 82.1%), while that for Lysed-RBC was >4.6/µL (area under the curve, 0.708; sensitivity, 82.4%; specificity, 56.0%; positive predictive value, 72.6%; and negative predictive value, 69.1%). Moreover, there was no significant difference in the sensitivity between the IgA nephropathy and non-IgA nephropathy groups (87.1 and 89.8% for UF-%sRBCs and 83.9 and 78.4% for Lysed-RBCs, respectively). In the NGN group, the cut-off values showed low sensitivity (56.0% for UF-%sRBCs and 44.0% for Lysed-RBCs). CONCLUSIONS: The RBC parameters of the UF-5000, specifically UF-%sRBCs and Lysed-RBCs, showed good cut-off values for the diagnosis of GN.

[Two Cases of Advanced and Recurrent Gastric Cancer with Long-Term Survival Due to Successful Chemotherapy].

Case 1: A 73-year-old man underwent total gastrectomy for residual gastric cancer, and final pathological diagnosis was pStage ⅠB. Adjuvant chemotherapy was not performed. CT findings showed multiple liver metastasis 16 months after procedure. S-1 and CDDP were administered for 28 months. Although chemotherapy regimen was changed to S-1, paclitaxel plus ramucirumab, nivolumab, irinotecan and S-1 plus oxaliplatin(SOX)after progression, he died 73 months after operation, and 57 months after recurrence. Case 2: A 72-year-old man was pointed out swelling of gastric lymph nodes in CT imaging. He was diagnosed as advanced gastric cancer with para-aortic lymph node metastasis by followed examination. S- 1 plus CDDP was administrated for 30 months. S-1 and SOX were administered after progressive findings, but he died 48 months after diagnosis. We report 2 cases of recurrent and advanced gastric cancer with long-term survival because of successful chemotherapy.

Reliability of serological tests for COVID-19: comparison of three immunochromatography test kits for SARS-CoV-2 antibodies.

BACKGROUND: Several immunochromatographic serological test kits have been developed to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies, but their relative performance and potential clinical utility is unclear. METHODS: Three commercially available serological test kits were evaluated using 99 serum samples collected from 29 patients diagnosed with coronavirus disease 2019 (COVID-19) and 100 serum samples collected from 100 healthy volunteers in 2017 as negative controls. RESULTS: The specificity of the IgM and IgG antibodies showed comparable results among the three immunochromatographic serological test kits. The specificity for IgM antibody was 98.0%, 98.0%, and 97.0%, and the specificity for IgG antibody was identical among the three kits (99.0%). The IgM antibody-positive rates of the three test kits for samples taken at the early stage of the disease (0-4 days after onset) were consistent with all three kits (18.2%); however, the IgM antibody-positive rates thereafter showed considerable differences among the kits, making it difficult to interpret the kinetics of IgM response against SARS-CoV-2. The IgG antibody-positive rates for samples taken after 13 days of onset were 100.0%, 97.6%, and 97.6%, respectively. CONCLUSION: There were large differences among the results of the three test kits. Only few cases showed positive results for IgM, suggesting that at least 2 of these kits used in this study were unsuitable for diagnosis of COVID-19. The IgG antibody was positive in almost all samples after 13 days of onset, suggesting that it may be useful for determining infections in the recent past.

Kynurenine produced by indoleamine 2,3-dioxygenase 2 exacerbates acute liver injury by carbon tetrachloride in mice.

Kynurenine (Kyn) plays an important role as an immune check-point molecule and regulates various immune responses through its aryl hydrocarbon receptor (Ahr). Kyn is synthesized by indoleamine 2,3-dioxygenase (Ido) and tryptophan 2,3-dioxygenase (Tdo). Ido contributes approximately 90% of tryptophan catabolism. Although Kyn is increased in various liver disorders, the roles of Kyn in liver injury are complicated because Ido1, Ido2, and Tdo are activated in different cell types. In this study, the roles of Ido2 in carbon tetrachloride (CCl4; 1 ml/kg, i.p.)-induced acute liver injury were examined using Ido2 knockout mice and Ido2 inhibitor. After CCl4 treatment, the ratio of Kyn to tryptophan and levels of Kyn in the liver were increased, accompanied by activation of Ahr-mediated signaling, as revealed by increased nuclear Ahr and Cyp1a1 mRNA. Knockout of Ido2 (Ido2-/-) and treatment with Ido2 inhibitor 1-methyl-D-tryptophan (D-1MT; 100 mg/kg, i.p.) attenuated CCl4-induced liver injury, with decreased induction of Ahr-mediated signaling. Administration of D-Kyn (100 mg/kg, i.p.) to Ido2-/- mice canceled the effect of Ido2 deficiency and exacerbated acute liver damage by CCl4 treatment. In addition, liver fibrosis induced by repeated CCl4 administration was suppressed in Ido2-/- mice. In conclusion, the action of Ido2 and Kyn in the liver may prevent severe hepatocellular damage and liver fibrosis.

Kynurenine plays an immunosuppressive role in 2,4,6-trinitrobenzene sulfate-induced colitis in mice.

BACKGROUND: Inflammatory bowel disease, such as Crohn's disease and ulcerative colitis, is characterized by chronic intestinal inflammation leading to intestinal mucosal damage. Inflammatory bowel disease causes dysregulation of mucosal T cell responses, especially the responses of CD4+ T cells. Previously, we demonstrated that indoleamine-2,3-dioxygenase plays an immunosuppressive role in 2,4,6-trinitrobenzene sulfate (TNBS)-induced colitis. Although indoleamine-2,3-dioxygenase exerts immunosuppressive effects by altering the local concentration of tryptophan (Trp) and immunomodulatory Trp metabolites, the specific changes in immune regulation during colitis caused by Trp metabolites and its related enzymes remain unclear. AIM: To investigate role of kynurenine 3-monooxygenase (KMO) in TNBS-induced colitis and involvement of Trp metabolites in maintenance of intestinal homeostasis. METHODS: Colitis was induced in eight-week-old male KMO+/+ or KMO-/- mice of C57BL/6N background using TNBS. Three days later, the colon was used for hematoxylin-eosin staining for histological grading, immunohistochemical or immunofluorescence staining for KMO, cytokines, and immune cells. Inflammatory and anti-inflammatory cytokines were measured using quantitative RT-PCR, and kynurenine (Kyn) pathway metabolites were measured by high-performance liquid chromatography. The cell proportions of colonic lamina propria and mesenteric lymph nodes were analyzed by flow cytometry. RESULTS: KMO expression levels in the colonic mononuclear phagocytes, including dendritic cells and macrophages increased upon TNBS induction. Notably, KMO deficiency reduced TNBS-induced colitis, resulting in an increased frequency of Foxp3+ regulatory T cells and increased mRNA and protein levels of anti-inflammatory cytokines, including transforming growth factor-ß and interleukin-10. CONCLUSION: Absence of KMO reduced TNBS-induced colitis via generation of Foxp3+ regulatory T cells by producing Kyn. Thus, Kyn may play a therapeutic role in colon protection during colitis.

[A Case of Metastatic Breast Cancer That Recovered from Diffuse Alveolar Damage Associated with Everolims].

A 68-year-old woman was receiving chemotherapy and endocrine therapy for right breast cancer postoperative pulmonary metastasis and local lymph node recurrence. However, she developed interstitial pneumonia 12 weeks after initiating treatment with everolimus and exemestane. Treatment with everolimus and exemestane was discontinued and steroid pulse therapy was initiated; however, she required ventilator management because of the severity of the pneumonia. The patient's condition improved 9 days after ventilator management. Everolimus-induced interstitial pneumonia is often mild, but it can be severe in rare cases. In our case, everolimus-induced diffuse alveoli damage was successfully treated with ventilator management.

Indoleamine 2,3-dioxygenase 2 depletion suppresses tumor growth in a mouse model of Lewis lung carcinoma.

Tryptophan metabolism is important to induce immune tolerance in tumors. To date, 3 types of tryptophan-metabolizing enzymes have been identified: indoleamine 2,3-dioxygenase 1 and 2 (IDO1 and IDO2) and tryptophan 2,3-dioxygenase 2. Numerous studies have focused on IDO1 as its expression is enhanced in various cancers. Recently, IDO2 has been identified as a tryptophan-metabolizing enzyme that is involved in several immune functions and expressed in cancers such as pancreatic cancer. However, the biological role of IDO2 in the induction of immune tolerance in tumors has not yet been reported. In the present study, we examined the effects of Ido2 depletion on tumor growth in a mouse model of Lewis lung carcinoma by using Ido2-knockout mice. Ido2-knockout mice had reduced tumor volumes compared to WT mice. Furthermore, Ido2 depletion altered the tumor microenvironment, such as tryptophan accumulation and kynurenine reduction, leading to enhancement of immune cell invasion. Finally, enzyme-linked immunospot assay revealed that Ido2 depletion enhanced γ-interferon secretion in the tumor. In conclusion, Ido2 is an important immune regulator in the tumor microenvironment. Our data indicate that IDO2 is a potential target for cancer treatment and drug development.

Development of renal failure in PargParp-1 null and Timm23 hypomorphic mice.

Poly(ADP-ribose) glycohydrolase (Parg) is a central enzyme for poly(ADP-ribose) degradation. We established a Parg+/- mice strain by deletion of a part of exon 1 and around 0.4-kb upstream of sequences of the Parg gene. Parg-/- embryos obtained by intercrossing the Parg+/- mice died in utero between 4.5 and 9.5 days postcoitum. We examined whether poly(ADP-ribose) polymerase-1 (Parp-1) deficiency could rescue embryonic lethality of Parg-/- mice. Parg-/-Parp-1-/- mice were born viable at a reduced frequency from the expected mendelian ratio in the intercross progeny of Parg+/-Parp-1-/- mice. The results suggest a possibility that the presence of Parp-1 is responsible for the lethality of Parg-/- embryos, and Parg molecules or Parg activity degrading poly(ADP-ribose) might be important for embryogenesis. In Parg-/-Parp-1-/- mice, Parg protein was not detected in various tissues, and the protein level of Timm23, a 5'-upstream gene of Parg, was reduced compared with that in Parg+/+Parp-1-/- mice. Parg-/-Parp-1-/- mice showed retarded growth compared with Parg+/+Parp-1-/- mice, and died within 3 months of age accompanied with severe renal failure. Glomerular sclerosis, tubular dilatation, and hyaline casts in the kidney were observed in Parg-/-Parp-1-/- mice. An increase in blood urea nitrogen (p < 0.05), a marked increase of albumin level in urine (p < 0.01) and its concomitant decrease in serum (p < 0.05) were also detected in Parg-/-Parp-1-/- mice compared with the Parg+/+Parp-1-/- counterpart. The results imply that the combined Parg and Parp-1 loss with a hypomorphic state of Timm23 leads to the development of severe renal failure.

[Analysis of Renal Toxicity of S-1 plus CDDP Regimen with Short Hydration for Outpatients with Gastric Cancer].

BACKGROUND: Although the S-1 plus CDDP(SP)regimen is the standard treatment for advanced gastric cancer, hydration and admission have been recommended after cisplatin has been administered. In this study, short hydration(SH)method was used and SP was administered in outpatient settings. We evaluated renal toxicity of cisplatin in the SH-SP regimen at our hospital. METHODS: Eleven of 16 patients(5 underwent only 1 course and so were excluded)received the SH-SP regimen between January 2012 and January 2018 to present and were included. Serum creatinine(Cr)and estimated glomerular filtration rate(eGFR)were used to assess renalfunction. RESULTS: Median course was 5. Rate of 5-course accomplishment was 72.7%. Grade 1 Cr elevation was observed in only 3 patients and there was no severe renal disorder. CONCLUSION: The SHSP regimen could be administered in outpatient settings and was considered safe as it did not cause renal toxicity.

[Clinical significance of hyaline casts in the new CKD risk classification (KDIGO 2009)].

Chronic kidney disease (CKD) significantly contributes to the increased number of dialysis patients with end stage renal disease. A new CKD risk classification (KDIGO 2009) established in 2011, which is defined by albuminuria and estimated glomerular filtration rate (eGFR) values, demonstrates the relative risks of CKD in great detail. In this study, we evaluated the clinical significance of urinary casts by categorizing a risk Group 1 to 5 according to the KDIGO 2009 classification. In the high risk CKD group (risk group 3 and over), we found a significantly higher number of patients who had > 100 hyaline casts/whole field (WF) in their urine than those that had < 100 hyaline casts/WF. Further, we determined the diagnostic accuracy for the high risk CKD group when the cutoff value for the number of hyaline casts was set at > or = 100 hyaline casts/WF (sensitivity: 44.7%, specificity: 96.5%). The eGFR value was significantly lower in the group with > or = 100 hyaline casts/WF, particularly in hypertensive patients, than that in the group with < 100 hyaline casts/ WF. Of interest is that the eGFR value was significantly lower in patients with 100-999 hyaline casts/WF and > or = 1,000 hyaline casts/WF than that in patients with < 100 hyaline casts/WF in A1 stage. Thus, our present study suggests that the presence of > or = 100 hyaline casts/WF indicates decreased eGFR, and the urinary casts counting may be important and useful for the screening and early detection of high-risk CKD.

Expression of E-cadherin and KRAS mutation may serve as biomarkers of cetuximab-based therapy in metastatic colorectal cancer.

Cetuximab (Cmab), a chimeric monoclonal antibody for targeting the epidermal growth factor receptor, has become one of the standard treatments for metastatic colorectal cancer (mCRC). However, only a small proportion of patients respond to Cmab, and it has been reported that KRAS mutation is a negative biomarker of response to Cmab therapy. The aim of this study was to detect additional biomarkers of response to Cmab therapy in patients with mCRC. We evaluated the effects of Cmab therapy in 36 patients with mCRC according to the Response Evaluation Criteria in Solid Tumors, and classified patients who achieved complete response, partial response or stable disease as responders, and patients who achieved progressive disease as non-responders. We retrospectively examined the difference between the two groups using KRAS analysis and immunohistochemistry to determine the expression of E-cadherin, p53 and Ki67. Nineteen patients were responders, while 17 patients were non-responders. KRAS status and expression of E-cadherin were significantly correlated with the effect of Cmab therapy. Moreover, the expression of E-cadherin was significantly correlated with the effect of Cmab therapy in KRAS wild-type patients. In KRAS mutant-type patients, the expression of E-cadherin did not significantly correlate with the effect of Cmab therapy, but all responders with KRAS mutant-type tumors expressed E-cadherin. Our results indicate that the expression of E-cadherin detected by immunohistochemistry may be a positive predictor of Cmab-based therapy in mCRC, and that a combination of E-cadherin immunohistochemistry and KRAS analysis may be a more sensitive biomarker than KRAS analysis alone.

[Three cases of giant rectal gastrointestinal stromal tumor].

The frequency of rectal gastrointestinal stromal tumor (GIST) is relatively low. We have experienced three cases of giant rectal GIST. Case 1 was treated with sunitinib after imatinib failed by Stevens-Johnson syndrome as neoadjuvant therapy. Case 2 was treated with imatinib as neoadjuvant therapy. These neoadjuvant therapies had no effect on tumor size. All patients underwent an abdominoperineal resection. The mean major axis was 11 .7 cm. Immnohistochemical staining showed that CD34 and KIT were positive. The term of follow-up is short, but no recurrences have been found in all cases. It has been reported that imatinib as neoadjuvant therapy is useful for radical resection in cases of giant rectal GIST. Furthermore, neoadjuvant therapy seems to be one of the treatment options for locally advanced rectal GIST. However, in cases of GIST patients not responding to imatinib, we should perform a surgical resection immediately.

[Pilot study of preoperative mFOLFOX6 chemotherapy for advanced rectal cancers which were difficult to ensure surgical margins].

Seven patients with rectal cancers which were difficult to ensure surgical margins for because of huge tumors(over 60mm in diameter), invasion to other organs, or severe nodal metastases, were treated with preoperative chemotherapy consisting of 2-10 courses of mFOLFOX6.The response rate was 85. 7%.Complete response was observed in one patient, and partial response was observed in 5 patients.Four to 5 weeks after chemotherapy, surgery was performed for all patients.Following surgical procedures, abdominoperineal resections were performed in 4 cases, low anterior resections in 3 cases, and removal of the liver metastases(not diagnosed preoperatively)in 2 cases.R0 resections were also performed in all patients. According to the histological regression grading of the resected specimens, one patient had a complete disappearance of tumor, 5 had grade 1a regression, and one had grade 1b regression.One of the 7 patients had recurrence at the lung. However, another patient survived without recurrence. In this study, preoperative mFOLFOX6 chemotherapy was expected to be an effective treatment for improving the curative resection rate of patients with tumors which were difficult to ensure surgical margins because of huge tumors, invasion to other organs, or severe nodal metastases.

In vitro assessment of anticryptosporidial efficacy and cytotoxicity of adenosine analogues using a SYBR Green real-time PCR method.

OBJECTIVES: The aims of this study were to provide a cost-effective and valuable method for evaluating drug efficacy against Cryptosporidium parvum using a quantitative SYBR Green real-time PCR (qPCR) and to assess the efficacy of adenosine analogues as drug templates. METHODS: C. parvum HNJ-1 strain growing in human ileocaecal adenocarcinoma cells was employed as an in vitro culture system. To normalize the DNA extraction efficiency, a specific plasmid was added to each sample before DNA purification; the genomic DNA of infected cells was quantified by qPCR using specific primers to confirm drug efficacy and cytotoxicity. To determine the mechanism of action, enzymatic inhibition analyses were conducted using C. parvum S-adenosyl-l-homocysteine hydrolase (CpSAHH) recombinant protein. RESULTS: The dose-dependent growth inhibition of C. parvum was confirmed; 50% effective concentrations of neplanocin A (NPA) and 2-fluoroadenosine (2FA) were 139 µM and 0.842 µM, respectively. Cytotoxicity evaluation showed that the 50% growth inhibition concentration of 2FA was 1.18 µM; NPA did not exhibit any cytotoxicity up to 200 µM. The screening system revealed the specific but marginal efficacy of NPA and showed 2FA to be cytotoxic. Recombinant CpSAHH inhibition analyses showed that NPA competitively inhibited CpSAHH activity (K(i )= 0.395 µM), whereas 2FA did not. CONCLUSIONS: This novel qPCR system confirmed not only drug efficacy against C. parvum but also cytotoxicity to host cells. Moreover, since the SYBR Green method is cost effective, it could therefore be used in a wide variety of clinical and research-oriented applications of Cryptosporidium analysis.

[A case of liver metastasis of pancreatic cancer that was resistant to S-1 and gemcitabine successfully treated by 5-FU and cisplatin hepatic arterial infusion].

We report a case of postoperative liver metastasis of pancreatic cancer that was resistant to S-1 and gemcitabine (GEM) successfully treated by hepatic arterial infusion of 5-fluorouracil (5-FU) and cisplatin (CDDP). A 70s woman was referred to our hospital for treatment of a pancreatic head cancer in November 2007. Pancreaticoduodenectomy with a regional lymphadectomy was performed in December 2007 and the pathological stage was Stage IVa. Adjuvant chemotherapy of UFT was administered one month after operation. However, a second chemotherapy of S-1 was administered because DUPAN-2 levels showed a high range 8 months after operation. Ten months after operation, abdominal computed tomography demonstrated a 2 cm tumor in the liver. Although, we performed a systemic GEM infusion and a combination of GEM and S-1, the liver metastasis had progressed. Then, hepatic arterial infusion (HAI) chemotherapy of 5-FU/CDDP was instituted weekly. This efficacy maintained a Partial Response from the start of HAI to the fifth month. She is alive to date, maintaining a stable tumor growth of 30 months after surgery. We suggest that HAI chemotherapy of 5-FU+CDDP might be an effective treatment to liver metastasis of pancreatic cancer and prolong prognosis of those patients.

Multiple-subgenotype infections of Giardia intestinalis detected in Palestinian clinical cases using a subcloning approach.

To evaluate the geographic distribution of Giardia intestinalis genotypes in Nablus, West Bank, Palestine, a genotyping study was performed using clinical fecal samples. Microscopic examination confirmed that 8 of 69 (11.6%) samples were G. intestinalis positive, and subsequent genotyping analyses targeting the small-subunit ribosomal RNA (18S rRNA) and glutamate dehydrogenase (GDH) genes revealed the G. intestinalis genotypes within the 8 samples. Of these 8 samples, 6 were clustered with assemblage A-II and the remaining 2 samples were clustered with assemblage B by 18S rRNA gene analysis; however, direct sequencing of the GDH gene segments from the latter 2 samples showed a mixed infection profile. To assess those samples, we employed a subcloning approach and successfully isolated 6 independent assemblage B subgenotypes. These partial GDH gene sequences (393 bp) had 15 single-nucleotide polymorphisms, all of which were synonymous transition substitutions at the third nucleotide position of codons. From the results, we concluded that the highly polymorphic gene loci such as GDH gene locus might provide us an opportunity to obtain a detailed molecular data even from the samples with multiple-subgenotype mixed infections. Therefore, subcloning approach is recommended in genotyping studies, especially in those conducted in giardiasis-endemic areas, where the repeated and cumulative infections could be commonly expected.