Purification of native histidine-rich protein 2 (nHRP2) from Plasmodium falciparum culture supernatant, infected RBCs, and parasite lysate.

BACKGROUND: Despite the widespread use of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), purified native HRP2 antigen is not standardly used in research applications or assessment of RDTs used in the field. METHODS: This report describes the purification of native HRP2 (nHRP2) from the HB3 Plasmodium falciparum culture strain. As this culture strain lacks pfhrp3 from its genome, it is an excellent source of HRP2 protein only and does not produce the closely-related HRP3. The nHRP2 protein was isolated from culture supernatant, infected red blood cells (iRBCs), and whole parasite lysate using nickel-metal chelate chromatography. Biochemical characterization of nHRP2 from HB3 culture was conducted by SDS-PAGE and western blotting, and nHRP2 was assayed by RDT, ELISA, and bead-based immunoassay. RESULTS: Purified nHRP2 was identified by SDS-PAGE and western blot as a - 60 kDa protein that bound anti-HRP-2 monoclonal antibodies. Mouse anti-HRP2 monoclonal antibody was found to produce high optical density readings between dilutions of 1:100 and 1:3,200 by ELISA with assay signal observed up to a 1:200,000 dilution. nHRP2 yield from HB3 culture by bead-based immunoassay revealed that both culture supernatant and iRBC lysate were practical sources of large quantities of this antigen, producing a total yield of 292.4 µg of nHRP2 from two pooled culture preparations. Assessment of nHRP2 recognition by RDTs revealed that Carestart Pf HRP2 and HRP2/pLDH RDTs detected purified nHRP2 when applied at concentrations between 20.6 and 2060 ng/mL, performing within a log-fold dilution of commercially-available recombinant HRP2. The band intensity observed for the nHRP2 dilutions was equivalent to that observed for P. falciparum culture strain dilutions of 3D7 and US06 F Nigeria XII between 12.5 and 1000 parasites/µL. CONCLUSIONS: Purified nHRP2 could be a valuable reagent for laboratory applications as well as assessment of new and existing RDTs prior to their use in clinical settings. These results establish that it is possible to extract microgram quantities of the native HRP2 antigen from HB3 culture and that this purified protein is well recognized by existing monoclonal antibody lines and RDTs.

HRP2 and HRP3 cross-reactivity and implications for HRP2-based RDT use in regions with Plasmodium falciparum hrp2 gene deletions.

BACKGROUND: The Plasmodium falciparum antigen histidine rich protein 2 (HRP2) is a preferred target for malaria rapid diagnostic tests (RDTs) because of its abundant production by the parasite and thermal stability. As a result, a majority of RDTs procured globally target this antigen. However, previous reports from South America and recent reports from sub-Saharan Africa and Asia indicate that certain P. falciparum parasites have deletions of the gene coding for HRP2. The HRP2 antigen is paralogous to another P. falciparum antigen HRP3 and some antibodies to HRP2 cross-react with HRP3. Multiple parasites have been described with deletions of one or both hrp2 and hrp3 genes. It is unclear how the various combinations of hrp2 and hrp3 deletion genotypes affect clinical sensitivity of HRP2-based RDTs. METHODS: Cross-reactivity between HRP2 and HRP3 was tested on malaria RDTs using culture-adapted P. falciparum parasites with both hrp2 and hrp3 intact or with one or both genes deleted. Ten-fold serial dilutions of four culture-adapted P. falciparum parasites [3D7 (hrp2+/hrp3+), Dd2 (hrp2-/hrp3+), HB3 (hrp2+/hrp3-) and 3BD5 (hrp2-/hrp3-)] ranging from 100,000 to 0.01 parasites/µL were prepared. HRP2, Plasmodium lactate dehydrogenase (pLDH) and aldolase concentrations were determined for the diluted samples using a multiplex bead assay. The samples were subsequently tested on three RDT products designed to detect P. falciparum by HRP2 alone or in combination with pLDH. RESULTS: At parasite densities of approximately 1000 parasites/µL, parasites that expressed either hrp2 or hrp3 were detected by all three RDTs. Multiplex based antigen measurement using HRP2- conjugated beads demonstrated higher antigen concentration when both hrp2 and hrp3 genes were intact (3D7 parasites, 47.9 ng/ml) compared to HB3 (3.02 ng/mL) and Dd2 (0.20 ng/mL) strains that had one gene deleted. 3D7 at 10 parasites/µL (0.45 ng/mL) was reactive on all three RDT products whereas none of the other parasites were reactive at that density. CONCLUSIONS: Above a certain antigen threshold, HRP3 cross-reactivity on HRP2-based RDTs is sufficient to mask the effects of deletions of hrp2 only. Studies of hrp2 deletion and its effects on HRP2-based RDTs must be studied alongside hrp3 deletions and include clinical sample reactivity on HRP2-based tests.

Impact of Plasmodium falciparum gene deletions on malaria rapid diagnostic test performance.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) have greatly improved access to diagnosis in endemic countries. Most RDTs detect Plasmodium falciparum histidine-rich protein 2 (HRP2), but their sensitivity is seriously threatened by the emergence of pfhrp2-deleted parasites. RDTs detecting P. falciparum or pan-lactate dehydrogenase (Pf- or pan-LDH) provide alternatives. The objective of this study was to systematically assess the performance of malaria RDTs against well-characterized pfhrp2-deleted P. falciparum parasites. METHODS: Thirty-two RDTs were tested against 100 wild-type clinical isolates (200 parasites/µL), and 40 samples from 10 culture-adapted and clinical isolates of pfhrp2-deleted parasites. Wild-type and pfhrp2-deleted parasites had comparable Pf-LDH concentrations. Pf-LDH-detecting RDTs were also tested against 18 clinical isolates at higher density (2,000 parasites/µL) lacking both pfhrp2 and pfhrp3. RESULTS: RDT positivity against pfhrp2-deleted parasites was highest (> 94%) for the two pan-LDH-only RDTs. The positivity rate for the nine Pf-LDH-detecting RDTs varied widely, with similar median positivity between double-deleted (pfhrp2/3 negative; 63.9%) and single-deleted (pfhrp2-negative/pfhrp3-positive; 59.1%) parasites, both lower than against wild-type P. falciparum (93.8%). Median positivity for HRP2-detecting RDTs against 22 single-deleted parasites was 69.9 and 35.2% for HRP2-only and HRP2-combination RDTs, respectively, compared to 96.0 and 92.5% for wild-type parasites. Eight of nine Pf-LDH RDTs detected all clinical, double-deleted samples at 2,000 parasites/µL. CONCLUSIONS: The pan-LDH-only RDTs evaluated performed well. Performance of Pf-LDH-detecting RDTs against wild-type P. falciparum does not necessarily predict performance against pfhrp2-deleted parasites. Furthermore, many, but not all HRP2-based RDTs, detect pfhrp2-negative/pfhrp3-positive samples, with implications for the HRP2-based RDT screening approach for detection and surveillance of HRP2-negative parasites.

Seroepidemiology of predominant norovirus strains circulating in Korea by using recombinant virus-like particle antigens.

An epidemiological survey on human norovirus (NoV)-associated gastroenteritis was conducted to clarify the prevalence of NoV infections in children and adults in Korea. Recombinant capsid proteins from three major NoV genotypes (GI-4, GII-3, and GII-4) were expressed using a baculovirus expression system, and the morphology and antigenicity of self-assembled virus-like particles were then confirmed by electron microscopy and Western blotting with a NoV-specific antibody. To determine seroprevalence, an enzyme-linked immunosorbent assay was performed to detect antibodies against virus-like particles antigen in 346 serum specimens collected from persons who visited five public heath care centers for regular physical examination in Jeollanam-do, Korea, between 2005 and 2006. The seroprevalence of immunoglobulin G antibodies against the GI-4, GII-3, and GII-4 NoV genotypes was 84.1%, 76.3%, and 94.5%, respectively. A rapid decrease in seroprevalence occurred after birth, with the lowest levels observed in the <23-month age group, and a steep increase in seroprevalence occurred in early childhood, reaching 60.5% for GI-4, 65.1% for GII-3, and 90.7% for GII-4 at age 2-5 years, and over 80% for all three genotypes in subjects aged 20 years or older. The seroprevalence of different NoV genotypes statistically differed across the age groups (p<0.01).

Occurrence of norovirus infections in asymptomatic food handlers in South Korea.

Prevalence of asymptomatic norovirus infection was investigated in food handlers in South Korea. Among 6,441 subjects, 66 (1.02%) had norovirus infections confirmed by reverse transcription (RT)-PCR (real time and nested). GII-12 and GII-4 were the prevalent genotypes. Our data suggest that infection of asymptomatic food handlers is an important transmission source in norovirus outbreaks.

Estimate Hearing Recovery Rate Using Statistical Analysis of Sudden Sensorineural Hearing Loss / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Sudden deafness is not rare but its pathophysiology has not been fully determined and treatment is controversial at present. There is much debate regarding the prognostic factors in sudden deafness patients. Various studies have been done to find out factors concerned with the recovery of sudden deafness, but there are still many unknown components as the probability of recovery from sudden deafness depends on many factors. The aim of this study is to identify an expectation of recovery by regression formula. SUBJECTS AND METHOD: We reviewed the records of 241 patients who were admitted for treatment between 2002 and 2005 under the initial diagnosis of idiopathic sudden hearing loss. The patients were treated with steroid, vasodilator and plasma expander. Improvement of hearing was measured by Siegel's criteria and correcting rate. Prognostic factors that might be associated with the degree of hearing recovery were classified and analyzed by the optimal scale regression analysis method using SPSS ver. 12.0. RESULTS: Siegel's criteria and correcting rate have statistical correlation with respect to age, initial hearing level, patterns of pure tone audiogram and duration of initial treatment from onset to hearing improvement after treatment. Vertigo is not associated with hearing improvement. CONCLUSION: The initial hearing level, patterns of pure tone audiogram and duration of initial treatment from onset are associated with the degree of hearing recovery. We produced a calculating model of hearing improvement based on factors affecting hearing improvement.

A Case of Cryptococcal Abscess Involving Deep Neck Space in an Immunocompetent Patient / 대한이비인후과학회지

The encapsulated yeast, Cryptococcus, has rapidly risen to a worldwide major opportunistic fungal pathogen. Infection with Cryptococcus neoformans usually occurs in immunocompromised host and but may occur also occur in immunocompetent patients. Cryptococccal infection commonly involves the lung or central nervous system; it can disseminate to any organ. However, Cryptococcal abscess in deep neck space is rare. We report an immunocompe-tent patient, who was 60-years-old with cryptococcal abscess in deep neck space. Incisional biopsy of the involved soft tissue revealed the presence of Cryptococcus neoformans. Abscess culture also yielded Cryptococcus. Surgical debriment and antifungal therapy resulted in cure.

Complete genome sequence and phylogenetic analysis of a recombinant Korean norovirus, CBNU1, recovered from a 2006 outbreak.

We have determined the complete nucleotide and deduced amino acid sequences of the RNA genome of CBNU1, a human norovirus (NoV) recovered from a 2006 outbreak in South Korea. The genome of 7547 nucleotides, excluding a 3'-poly(A) tail of 11-105 nucleotides, encodes three overlapping open reading frames (ORFs): ORF1 (nucleotides 5-5104), ORF2 (nucleotides 5085-6731), and ORF3 (nucleotides 6731-7495). In a comparison to 108 other currently available completely sequenced NoVs representing all five genogroups (GI-GV) except GIV, the CBNU1 strain was highly similar to GII.3 NoVs. Multiple sequence alignments of the completely sequenced NoV genomes revealed five hypervariable regions throughout their genomes: two in ORF1, one in ORF2, and two in ORF3. At both the nucleotide and amino acid levels, genome-based phylogenetic analyses invariably showed that the CBNU1 strain was most closely related to three GII.3 NoVs: the American Texas/TCH04-577 and the two Japanese Saitama U18 and Saitama U201 strains; furthermore, these genome-based phylogenetic topologies corresponded most closely to those based on the ORF2 genes, as compared to those based on the ORF1 and ORF3 genes. Subsequent ORF2-based phylogenetic analyses of a selection of 126 other NoVs representing all 19 GII genotypes, in combination with genome-based Simplot analyses, showed that the CBNU1 strain was a recombinant GII.3 NoV with a breakpoint at the ORF1/ORF2 junction between two putative parent-like strains, Guangzhou/NVgz01 and Texas/TCH04-577. Overall, the CBNU1 strain represents the first Korean human NoV whose genome has been completely sequenced and for which its relationship with a large panel of genetically diverse NoVs has been extensively characterized.