Predictive model for bacterial late-onset neonatal sepsis in a tertiary care hospital in Thailand.

BACKGROUND: Early diagnosis of neonatal sepsis is essential to prevent severe complications and avoid unnecessary use of antibiotics. The mortality of neonatal sepsis is over 18%in many countries. This study aimed to develop a predictive model for the diagnosis of bacterial late-onset neonatal sepsis. METHODS: A case-control study was conducted at Queen Sirikit National Institute of Child Health, Bangkok, Thailand. Data were derived from the medical records of 52 sepsis cases and 156 non-sepsis controls. Only proven bacterial neonatal sepsis cases were included in the sepsis group. The non-sepsis group consisted of neonates without any infection. Potential predictors consisted of risk factors, clinical conditions, laboratory data, and treatment modalities. The model was developed based on multiple logistic regression analysis. RESULTS: The incidence of late proven neonatal sepsis was 1.46%. The model had 6 significant variables: poor feeding, abnormal heart rate (outside the range 100-180 x/min), abnormal temperature (outside the range 36o-37.9 °C), abnormal oxygen saturation, abnormal leucocytes (according to Manroe's criteria by age), and abnormal pH (outside the range 7.27-7.45). The area below the Receiver Operating Characteristics (ROC) curve was 95.5%. The score had a sensitivity of 88.5% and specificity of 90.4%. CONCLUSION: A predictive model and a scoring system were developed for proven bacterial late-onset neonatal sepsis. This simpler tool is expected to somewhat replace microbiological culture, especially in resource-limited settings.

Immunogenicity and safety of inactivated chromatographically purified Vero cell-derived Japanese encephalitis vaccine in Thai children.

Inactivated mouse-brain-derived Japanese encephalitis vaccine has a worrisome safety profile and the live attenuated vaccine is unsuitable in immunodeficiency. This study aimed to evaluate the immunogenicity and safety of an inactivated chromatographically purified Vero-cell-derived JE vaccine (CVI-JE, Beijing P-3 strain) in children. 152 healthy Thai children, with an average (SD) age of 14.4 (3.8) months, received 3 doses of CVI-JE on days 0, 7-28, and one year. Homologous JE neutralizing antibody titers (NT) were measured. All subjects had seroprotection [geometric mean titer (GMT) 150] 28 days' post 2nd vaccination. The seroprotection rates at 1 year after primary series and and 1 month after the booster were 89.3% (GMT 49) and 100% (GMT 621), respectively. Local and systemic reactions-fever (17.6%), vomiting (8%), and poor appetite (5.3%)-were noted within 28 days' post-vaccination. All these symptoms were self-limited. CONCLUSIONS: CVI-JE is safe, immunogenic, and provided high NT.

Severe vivax malaria: a systematic review and meta-analysis of clinical studies since 1900.

BACKGROUND: Malaria caused by Plasmodium vivax was long considered to have a low mortality, but recent reports from some geographical areas suggest that severe and complicated vivax malaria may be more common than previously thought. METHODS: The primary objective of this systematic review and meta-analysis was to describe the reported clinical characteristics and the geographical variation in prevalence of reported severe vivax malaria and its change over time derived from English-language articles published since 1900. Medline and Scopus databases were searched for original papers on severe vivax malaria, using as inclusion criteria modified 2010 WHO criteria for the diagnosis of severe falciparum malaria. Articles before 1949 were identified through reference lists in journals, textbooks, and personal collections of colleagues. RESULTS: A total of 77 studies with reported severe vivax malaria and 63 studies with no reported severe vivax malaria (totaling 46,411 and 6,753 vivax malaria patients, respectively) were included. The 77 studies with reported severe vivax malaria were mainly from India (n = 33), USA (n = 8), Indonesia (n = 6), and Pakistan (n = 6). Vivax endemic countries not reporting severe vivax malaria beyond individual case reports included: the Greater Mekong Sub-region, China, North Korea, Bangladesh, Afghanistan, Middle East (except Qatar), the horn of Africa, and Madagascar. Only 17/77 reports were from before 2000. Vivax mono-infection was confirmed by PCR in 14 studies and co-morbidities were ruled out in 23 studies. Among the 77 studies reporting severe vivax malaria, severe thrombocytopenia (<50,000/mm3) was the most common "severe" manifestation (888/45,775 with pooled prevalence of 8.6%). The case fatality was 0.3% (353/46,411). Severity syndromes varied widely between different geographical areas, with severe anaemia being most prominent in areas of high transmission and chloroquine resistance. CONCLUSION: Plasmodium vivax can cause severe and even fatal disease, but there is a recent increase in reports over the past 15 years with larger series restricted to a limited number of geographical areas. The biological basis of these variations is currently not known. More detailed epidemiological studies are needed which dissociate causation from association to refine the definition and estimate the prevalence of severe vivax malaria.

A randomized comparison of dihydroartemisinin-piperaquine and artesunate-amodiaquine combined with primaquine for radical treatment of vivax malaria in Sumatera, Indonesia.

BACKGROUND: A high prevalence of chloroquine-resistant Plasmodium vivax in Indonesia has shifted first-line treatment to artemisinin-based combination therapies, combined with primaquine (PQ) for radical cure. Which combination is most effective and safe remains to be established. METHODS: We conducted a prospective open-label randomized comparison of 14 days of PQ (0.25 mg base/kg) plus either artesunate-amodiaquine (AAQ + PQ) or dihydroartemisinin-piperaquine (DHP + PQ) for the treatment of uncomplicated monoinfection P. vivax malaria in North Sumatera, Indonesia. Patients were randomized and treatments were given without prior testing for G6PD status. The primary outcome was parasitological failure at day 42. Patients were followed up to 1 year. RESULTS: Between December 2010 and April 2012, 331 patients were included. After treatment with AAQ + PQ, recurrent infection occurred in 0 of 167 patients within 42 days and in 15 of 130 (11.5%; 95% confidence interval [CI], 6.6%-18.3%) within a year. With DHP + PQ, this was 1 of 164 (0.6%; 95% CI, 0.01%-3.4%) and 13 of 143 (9.1%; 95% CI, 4.9%-15.0%), respectively (P > .2). Intravascular hemolysis occurred in 5 patients, of which 3 males were hemizygous for the G6PD-Mahidol mutation. Minor adverse events were more frequent with AAQ + PQ. CONCLUSIONS: In North Sumatera, Indonesia, AAQ and DHP, both combined with PQ, were effective for blood-stage parasite clearance of uncomplicated P. vivax malaria. Both treatments were safe, but DHP + PQ was better tolerated. CLINICAL TRIALS REGISTRATION: NCT01288820.

Differences in clinical features between children and adults with dengue hemorrhagic fever/dengue shock syndrome.

This retrospective study was conducted to assess the differences in clinical features between children and adults with dengue hemorrhagic fever/ dengue shock syndrome (DHF/DSS) admitted to Ratchaburi Hospital, Ratchaburi Province, Thailand. A total of 273 patients with DHF/DSS admitted to Ratchaburi Hospital during January 2007 to May 2008 were included in the study. The median age (range) of studied subjects was 16 years (6 months to 62 years) and the ratio of adults to children was 1.6:1. Forty-eight percent of subjects were 16-30 years old. The common signs, symptoms and clinical features were: nausea/vomiting (74.0%), a positive tourniquet test (73.0%), anorexia (67.0%), hemoconcentration (58.0%), headache (54.0%), abdominal tenderness (43.0%), myalgia (39.0%) and pleural effusion (20.0%). Children had anorexia, a positive tourniquet test, abdominal tenderness and a convalescent rash more frequently than adults. Children also had significantly more prominent plasma leakage as shown by lower serum albumin and sodium and a higher prevalence of pleural effusion, ascites and shock. Although not statistically significant, the prevalence of bleeding in children was higher than in adults but more adults needed blood transfusion. This study provides additional insight into the clinical picture of DHF/DSS in adults and children and may be beneficial for clinicians caring for these adults and children.

The Plasmodium falciparum erythrocyte invasion ligand Pfrh4 as a target of functional and protective human antibodies against malaria.

BACKGROUND: Acquired antibodies are important in human immunity to malaria, but key targets remain largely unknown. Plasmodium falciparum reticulocyte-binding-homologue-4 (PfRh4) is important for invasion of human erythrocytes and may therefore be a target of protective immunity. METHODS: IgG and IgG subclass-specific responses against different regions of PfRh4 were determined in a longitudinal cohort of 206 children in Papua New Guinea (PNG). Human PfRh4 antibodies were tested for functional invasion-inhibitory activity, and expression of PfRh4 by P. falciparum isolates and sequence polymorphisms were determined. RESULTS: Antibodies to PfRh4 were acquired by children exposed to P. falciparum malaria, were predominantly comprised of IgG1 and IgG3 subclasses, and were associated with increasing age and active parasitemia. High levels of antibodies, particularly IgG3, were strongly predictive of protection against clinical malaria and high-density parasitemia. Human affinity-purified antibodies to the binding region of PfRh4 effectively inhibited erythrocyte invasion by P. falciparum merozoites and antibody levels in protected children were at functionally-active concentrations. Although expression of PfRh4 can vary, PfRh4 protein was expressed by most isolates derived from the cohort and showed limited sequence polymorphism. CONCLUSIONS: Evidence suggests that PfRh4 is a target of antibodies that contribute to protective immunity to malaria by inhibiting erythrocyte invasion and preventing high density parasitemia. These findings advance our understanding of the targets and mechanisms of human immunity and evaluating the potential of PfRh4 as a component of candidate malaria vaccines.

Dengue infection in children in Ratchaburi, Thailand: a cohort study. II. Clinical manifestations.

BACKGROUND: Dengue infection is one of the most important mosquito-borne diseases. More data regarding the disease burden and the prevalence of each clinical spectrum among symptomatic infections and the clinical manifestations are needed. This study aims to describe the incidence and clinical manifestations of symptomatic dengue infection in Thai children during 2006 through 2008. STUDY DESIGN: This study is a school-based prospective open cohort study with a 9,448 person-year follow-up in children aged 3-14 years. Active surveillance for febrile illnesses was done in the studied subjects. Subjects who had febrile illness were asked to visit the study hospital for clinical and laboratory evaluation, treatment, and serological tests for dengue infection. The clinical data from medical records, diary cards, and data collection forms were collected and analyzed. RESULTS: Dengue infections were the causes of 12.1% of febrile illnesses attending the hospital, including undifferentiated fever (UF) (49.8%), dengue fever (DF) (39.3%) and dengue hemorrhagic fever (DHF) (10.9%). Headache, anorexia, nausea/vomiting and myalgia were common symptoms occurring in more than half of the patients. The more severe dengue spectrum (i.e., DHF) had higher temperature, higher prevalence of nausea/vomiting, abdominal pain, rash, diarrhea, petechiae, hepatomegaly and lower platelet count. DHF cases also had significantly higher prevalence of anorexia, nausea/vomiting and abdominal pain during day 3-6 and diarrhea during day 4-6 of illness. The absence of nausea/vomiting, abdominal pain, diarrhea, petechiae, hepatomegaly and positive tourniquet test may predict non-DHF. CONCLUSION: Among symptomatic dengue infection, UF is most common followed by DF and DHF. Some clinical manifestations may be useful to predict the more severe disease (i.e., DHF). This study presents additional information in the clinical spectra of symptomatic dengue infection.

Comparison of real-time SYBR green dengue assay with real-time taqman RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection.

BACKGROUND: Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman(®) assay and conventional nested PCR assay. AIMS: To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conventional nested PCR (modified Lanciotti). MATERIALS AND METHODS: Eight cultured virus strains were diluted in tenth dilution down to undetectable level by the PCR to optimize the primer, temperature (annealing, and extension and to detect the limit of detection of the assay. Hundred and ninety three ELISA and PCR proved dengue clinical samples were tested with real time SYBR(®) Green assay, real time Taqman(®) assay to compare the sensitivity and specificity. RESULTS: Sensitivity and specificity of real time SYBR® green dengue assay (84% and 66%, respectively) was almost comparable to those (81% and 74%) of Taqman real time PCR dengue assay. Real time SYBR(®) green RT-PCR was equally sensitive in primary and secondary infection while real time Taqman was less sensitive in the secondary infection. Sensitivity of real time Taqman on DENV3 (87%) was equal to SYBR green real time PCR dengue assay. CONCLUSION: We developed low cost rapid diagnostic SYBR green dengue assay. Further study is needed to make duplex primer assay for the serotyping of dengue virus.

Evidence that the erythrocyte invasion ligand PfRh2 is a target of protective immunity against Plasmodium falciparum malaria.

Abs targeting blood-stage Ags of Plasmodium falciparum are important in acquired immunity to malaria, but major targets remain unclear. The P. falciparum reticulocyte-binding homologs (PfRh) are key ligands used by merozoites during invasion of erythrocytes. PfRh2a and PfRh2b are functionally important members of this family and may be targets of protective immunity, but their potential role in human immunity has not been examined. We expressed eight recombinant proteins covering the entire PfRh2 common region, as well as PfRh2a- and PfRh2b-specific regions. Abs were measured among a cohort of 206 Papua New Guinean children who were followed prospectively for 6 mo for reinfection and malaria. At baseline, Abs were associated with increasing age and active infection. High levels of IgG to all PfRh2 protein constructs were strongly associated with protection from symptomatic malaria and high-density parasitemia. The predominant IgG subclasses were IgG1 and IgG3, with little IgG2 and IgG4 detected. To further understand the significance of PfRh2 as an immune target, we analyzed PfRh2 sequences and found that polymorphisms are concentrated in an N-terminal region of the protein and seem to be under diversifying selection, suggesting immune pressure. Cluster analysis arranged the sequences into two main groups, suggesting that many of the haplotypes identified may be antigenically similar. These findings provide evidence suggesting that PfRh2 is an important target of protective immunity in humans and that Abs act by controlling blood-stage parasitemia and support its potential for vaccine development.

Case-control approach to identify Plasmodium falciparum polymorphisms associated with severe malaria.

BACKGROUND: Studies to identify phenotypically-associated polymorphisms in the Plasmodium falciparum 23 Mb genome will require a dense array of marker loci. It was considered promising to undertake initial allelic association studies to prospect for virulence polymorphisms in Thailand, as the low endemicity would allow higher levels of linkage disequilibrium (LD) than would exist in more highly endemic areas. METHODOLOGY/PRINCIPAL FINDINGS: Assessment of LD was first made with 11 microsatellite loci widely dispersed in the parasite genome, and 16 microsatellite loci covering a approximately 140 kb region of chromosome 2 (an arbitrarily representative non-telomeric part of the genome), in a sample of 100 P. falciparum isolates. The dispersed loci showed minimal LD (Index of Association, I(S) (A) = 0.013, P = 0.10), while those on chromosome 2 showed significant LD values mostly between loci <5 kb apart. A disease association study was then performed comparing parasites in 113 severe malaria cases and 245 mild malaria controls. Genotyping was performed on almost all polymorphisms in the binding domains of three erythrocyte binding antigens (eba175, eba140 and eba181), and repeat sequence polymorphisms approximately 2 kb apart in each of three reticulocyte binding homologues (Rh1, Rh2a/b, and Rh4). Differences between cases and controls were seen for (i) codons 388-90 in eba175, and (ii) a repeat sequence centred on Rh1 codon 667. CONCLUSIONS/SIGNIFICANCE: Allelic association studies on P. falciparum require dense genotypic markers, even in a population of only moderate endemicity that has more extensive LD than highly endemic populations. Disease-associated polymorphisms in the eba175 and Rh1 genes encode differences in the middle of previously characterised erythrocyte binding domains, marking these for further investigation.

Contrasting signatures of selection on the Plasmodium falciparum erythrocyte binding antigen gene family.

Erythrocyte binding antigens of Plasmodium falciparum are involved in erythrocyte invasion, and may be targets of acquired immunity. Of the five eba genes, protein products have been detected for eba-175, eba-181 and eba-140, but not for psieba-165 or ebl-1, providing opportunity for comparative analysis of genetic variation to identify selection. Region II of each of these genes was sequenced from a cross-sectional sample of parasites in an endemic Kenyan population, and the frequency distributions of polymorphisms analysed. A positive value of Tajima's D was observed for eba-175 (D=1.13) indicating an excess of intermediate frequency polymorphisms, while all other genes had negative values, the most negative being ebl-1 (D=-2.35) followed by psieba-165 (D=-1.79). The eba-175 and ebl-1 genes were then studied in a sample of parasites from Thailand, for which a positive Tajima's D value was again observed for eba-175 (D=1.79), and a negative value for ebl-1 (D=-1.85). This indicates that eba-175 is under balancing selection in each population, in strong contrast to the other members of the gene family, particularly ebl-1 and psieba-165 that may have been under recent directional selection. Population expansion simulations were performed under a neutral model, further supporting the departures from neutrality of these genes.

Allele frequency-based analyses robustly map sequence sites under balancing selection in a malaria vaccine candidate antigen.

The Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading candidate for a malaria vaccine. Here, within-population analyses of alleles from 50 Thai P. falciparum isolates yield significant evidence for balancing selection on polymorphisms within the disulfide-bonded domains I and III of the surface accessible ectodomain of AMA1, a result very similar to that seen previously in a Nigerian population. Studying the frequency of nucleotide polymorphisms in both populations shows that the between-population component of variance (F(ST)) is significantly lower in domains I and III compared to the intervening domain II and compared to 11 unlinked microsatellite loci. A nucleotide site-by-site analysis shows that sites with exceptionally high or low F(ST) values cluster significantly into serial runs, with four runs of low values in domain I and one in domain III. These runs may map the sequences that are consistently under the strongest balancing selection from naturally acquired immune responses.

Safety and immunogenicity of tetravalent live-attenuated dengue vaccines in Thai adult volunteers: role of serotype concentration, ratio, and multiple doses.

Dengue fever, caused by four serotypes of a mosquito-borne virus, is a growing problem in tropical countries. Currently, there is no treatment or vaccine. We evaluated safety and immunogenicity of two doses, given six months apart, of seven formulations of dengue tetravalent live-attenuated vaccine (containing different concentrations of the component viruses) versus placebo in 59 flavivirus-seronegative Thai adults. The first dose was the more reactogenic. Most volunteers experienced clinically moderate fever, headache, myalgia, eye pain or rash 7-11 days after injection, generally lasting three days or less. Modest decreases in platelets and neutrophils were observed. After one dose, 58% of dengue recipients seroconverted (neutralizing antibody level > or = 1:10) against > or = 3 serotypes; 35% seroconverted against all four. After the second dose, seroconversion was 76% and 71%, respectively. All subjects seroconverted to serotype 3 after one dose. Serotype 4 elicited the lowest primary response but the highest increase in seroconversion after the second dose.