Natural selection of K13 mutants of Plasmodium falciparum in response to artemisinin combination therapies in Thailand.

Resistance of Plasmodium falciparum to artemisinin combination therapy (ACT) in Southeast Asia can have a devastating impact on chemotherapy and control measures. In this study, the evolution of artemisinin-resistant P. falciparum in Thailand was assessed by exploring mutations in the K13 locus believed to confer drug resistance phenotype. P. falciparum-infected blood samples were obtained from patients in eight provinces of Thailand over two decades (1991-2014; n = 904). Analysis of the K13 gene was performed by either sequencing the complete coding region (n = 259) or mutation-specific PCR-restriction fragment length polymorphism method (n = 645). K13 mutations related to artesunate resistance were detected in isolates from Trat province bordering Cambodia in 1991, about 4 years preceding widespread deployment of ACT in Thailand and increased in frequency over time. Nonsynonymous nucleotide diversity exceeded synonymous nucleotide diversity in the propeller region of the K13 gene, supporting the hypothesis that this diversity was driven by natural selection. No single mutant appeared to be favoured in every population, and propeller-region mutants were rarely observed in linkage with each other in the same haplotype. On the other hand, there was a highly significant association between the occurrence of a propeller mutant and the insertion of two or three asparagines after residue 139 of K13. Whether this insertion plays a compensatory role for deleterious effects of propeller mutants on the function of the K13 protein requires further investigation. However, modification of duration of ACT from 2-day to 3-day regimens in 2008 throughout the country does not halt the increase in frequency of mutants conferring artemisinin resistance phenotype.

Mitochondrial respiratory chain dysfunction in various neuromuscular diseases.

The mitochondrial respiratory chain function and the occurrence of mitochondrial respiratory chain dysfunction were determined in various neuromuscular diseases. The mitochondrial complexes I-V and citrate synthase in the skeletal muscle taken from 75 orthopaedic surgical patients excluding neuromuscular diseases (control subjects) and 26 patients with various neuromuscular diseases (7 patients with Duchenne muscular dystrophy, 3 patients with spinal muscular atrophy, 6 patients with mitochondrial diseases, 7 patients with type II fibre atrophy and 3 patients with neuropathy) were assayed. Of 26 patients, results of analysis of 3 patients (1 Duchenne muscular dystrophy, 1 spinal muscular atrophy and 1 type II fibre atrophy) were excluded because the citrate synthase activities in their muscle homogenate were less than third percentile of the normal controls. As compared to the control subjects by using Student's t-test, all studied groups of patients had significantly lower activities of more than one or two mitochondrial complexes (p<0.05). However, a significantly higher activity of mitochondrial complex I was observed in patients with mitochondrial diseases (p<0.05). These findings will require further study to elucidate the pathogenesis and role of secondary mitochondrial respiratory chain dysfunction in such neuromuscular diseases.

Prevalence of climacteric symptoms according to years after menopause.

OBJECTIVE: To study the prevalence of menopausal symptoms of women attending the menopause and gynecology clinics at Chulalongkorn Hospital. STUDY DESIGN: A descriptive study was conducted at the menopause and gynecology clinics at Chulalongkorn Hospital. After inclusion and exclusion were done, four hundred and twenty seven participants with premenopause, perimenopause and postmenopause were studied. All the women were classified into seven groups of premenopause, perimenopause and one, two, three, four and > or =five years after menopause. The interview was performed by well-trained social workers using standardized questionnaires. RESULTS: The average age at menopause of the postmenopausal women was 49.46 + 3.30 years. Prevalence of vasomotor symptoms eg. hot flushes in premenopause, perimenopause and one, two, three, four and > or =five years after menopause were 4.4 per cent, 25 per cent, 27.3 per cent, 38.8 per cent, 40 per cent, 11.1 per cent and 10.3 per cent, respectively. Prevalence of psychological symptoms eg. moodiness were 26.5 per cent, 25 per cent, 54.6 per cent, 38.7 per cent, 32.2 per cent, 11.2 per cent and 11.8 per cent, respectively. But the prevalence of headache in this category was 29.4 per cent, 23.3 per cent, 23.7 per cent, 22.6 per cent, 25.0 per cent, 11.1 per cent and 13.2 per cent, respectively. Prevalence of urinary symptoms seemed to increase continuously after menopause. Prevalence of genital symptoms eg. vaginal dryness were 5.9 per cent, 13.3 per cent, 25.5 per cent, 25.8 per cent, 15.0 per cent, 16.7 per cent and 20.6 per cent, respectively. Prevalence of other symptoms eg. muscle and joint pain were 22.1 per cent, 43.3 per cent, 56.4 per cent, 58.0 per cent, 45.0 per cent, 27.8 per cent and 28.0 per cent, respectively. CONCLUSION: The prevalence of menopausal symptoms in this postmenopausal group appeared to increase during the first and second years after menopause and tended to decrease afterwards. The prevalence of other symptoms eg. dry eyes and headache appeared to be unchanged after menopause.

Uterine perforation with Lippes loop intrauterine device-associated actinomycosis: a case report and review of the literature.

A case of a 67-year-old postmenopausal woman, gravida 2, para 2, with an uterine perforation from actinomycotic infection with Lippes loop IUD is reported. She had the Lippes loop IUD inserted for 35 years, and had never had any pelvic examination nor Papanicolaou smear. She presented with acute abdominal pain. The clinical picture mimicked peptic ulcer perforation. The woman underwent laparotomy and exudative fluid was discovered in the abdominal cavity with the tip of the Lippes loop IUD at one of the two small holes of the uterine fundus. Total abdominal hysterectomy with bilateral salpingo-oophorectomy was performed. The postoperative microscopic pathological report demonstrated characteristics of actinomycosis. She was treated with parenteral high-dose penicillin for 4 weeks followed by oral penicillin for 6 months. The woman had an uneventful recovery. To our knowledge, this is the first case report of uterine perforation due to Lippes loop IUD-associated actinomycotic infection.

Rosetting of Plasmodium falciparum required multiple components of the uninfected erythrocytes.

The mechanism of rosette formation of uninfected erythrocytes with Plasmodium falciparum-infected erythrocytes is rarely described. In this study, rosetting of uninfected normal erythrocytes with infected erythrocytes significantly reduced after treatment of the uninfected erythrocytes with neuraminidase. In contrast, the rosetting property of the infected erythrocytes was abolished by trypsinization but not by neuraminidase. The in vitro rosetting model showed that uninfected thalassemic erythrocytes poorly formed rosettes with infected normal erythrocytes when compared with normal erythrocytes of the same blood group. A rosetting parasite clone showed significant reduction in rosetting with thalassemic erythrocytes of all blood groups, however, this reduction was not obvious when the wild P. falciparum isolates were studied. These results suggest that while parasites from a single clone can rosette with uninfected erythrocytes via carbohydrate component, there is more than one type of receptor on uninfected erythrocytes involved in rosette formation with the heterogeneous populations of the wild P. falciparum isolates.

Population genetic data on loci LDLR, GYPA, HBGG, D7S8 and GC in the Bangkok population compared with rural Thais from Trat province.

Prior to the introduction of any DNA marker as a tool for person identification and paternity test in certain ethnic groups, a population genetic database should be constructed. Using multiplex primers in single tube polymerase chain amplification, 5 loci of unrelated genes in the PM Amplitype kit (Perkin Elmer) were studied in two Thai population groups: 228 DNA samples were extracted from blood collected at the Borai rural area in Trat province; another 123 DNA samples were collected at the outpatient clinic, Department of Forensic Medicine, King Chulalongkorn Memorial Hospital, Bangkok. Analysis of alleles and genotypes was performed after reversed dot blot hybridization of PCR products to allelic sequence specific probes immobilized on the membrane strip followed by nonradioisotopic detection according to the manufacturer's protocol. Population genetic statistic parameters including discrimination power (DP), the probability of matching (PM), power of exclusion for trio (PE trio) and typical paternity index (PI typical) were computed. Both Thai population groups showed no significant deviation from the Hardy Weinberg Expectation (HWE). The combined DP of all 5 loci in the PM Amplitype markers was 0.993636 for rural Thais and 0.994409 for Thais from Bangkok. The combined PM for rural Thais and those living in Bangkok was 0.006364 and 0.005591, respectively. The combined PE trio was 0.696825 and 0.698875 in both Thai population groups and the combined PI typical values were < 1.0. In conclusion, person identification using PM Amplitype DNA markers was efficient and satisfactory within certain limits. Hence, the application of PM Amplitype DNA markers for paternity tests should be cautiously considered and applied in combination with other parameters.

The variation of Pf155/RESA gene in field isolates of Plasmodium falciparum from Thailand.

Pf155/RESA, an antigen found on the surface of Plasmodium falciparum red blood cell membrane was once a proposed malarial vaccine candidate. The complete sequence of Pf155/RESA gene from one strain and partial sequence from two other isolates revealed that the gene is well conserved. But polymorphism of other antigenic encoded regions occurs with high frequency among isolates especially those collected from the field. Using solid phase sequencing technique, the nested PCR products of upstream 3' repeated region of exon 2 RESA gene were studied in 150 P. falciparum isolates. Of which 117 isolates were directly collected from the field and sequenced. Other samples studied include clones and cryopreserved of previously cultured isolates. The resulting sequences are compared with previously existing data of F32 (Tanzania) and FC27 (Papua New Guinea) designated as allelic type I and II respectively. Sequence analysis of the 150 P. falciparum showed that the amplified region of RESA gene was highly variable with substitution ranging from one to six bases and these allelic variables can be divided into 10 types. The frequency of type I(F32) occurrence is 70.86 percent, type III 13.38 per cent and 0.78 percent to 5.51 per cent for others. As a result of allelic polymorphism, the amino acid sequence is highly variable and this may cause Pf155/RESA to be an inefficient antigen.

Multiplex PCR to detect the dystrophin gene deletion in Thai patients.

We have demonstrated the usefulness of the multiplex PCR to directly detect the dystrophin gene mutation. Prenatal diagnosis and confirmation of clinical diagnosis of DMD/BMD via non invasive technique are now possible. Nine DMD and one BMD patients were tested. Five DMD patients demonstrated deletion. Thus, this multiplex PCR could detect deletion in approximately 50 per cent of DMD/BMD Thai patients. Eighty per cent of the deletions were in the distal part, whereas, 20 per cent were in the proximal part. We are planning to establish other molecular techniques such as linkage analysis, cDNA hybridization and immunostaining of dystrophin protein to improve a mode of diagnosis and management of DMD/BMD patients in the Thai community.

Alteration in cytoadherence and rosetting of Plasmodium falciparum-infected thalassemic red blood cells.

Hemoglobinopathies have a protective role in malaria that appears to be related to alterations in red blood cell (RBC) properties. Thalassemic RBCs infected with Plasmodium falciparum showed greatly reduced cytoadherence and rosetting properties as well as impaired growth and multiplication. A significant decrease in the levels of falciparum antigens associated with the membrane of infected beta-thalassemic RBCs was observed at trophozoite/schizont stage, but not young ring stage. This reduction was shown when a cytoadherence inhibitory monoclonal antibody, but not a noninhibitory pooled immune serum, was used. These observations suggest that protection against malaria in thalassemia is caused by both reduced parasitemias and altered adherence properties of the infected thalassemic RBCs that promote enhanced clearance of the parasite from the circulation.

Plasma C3c in immune-mediated neurological diseases: a preliminary report.

Plasma C3c levels were examined in 56 patients with immune (27) and non-immune (29) mediated neurological diseases by crossed immunoelectrophoresis. Plasma samples were collected during the active phase of illness in both groups, usually within 7 days of admission. 11 patients (4 Guillain-Barré Syndrome-GBS, 3 chronic inflammatory demyelinating polyneuropathy-CIDP, 4 myasthenia gravis-MG) had their plasma saved sequentially during the active and the recovery phase. Plasma C3c levels were elevated in the group with immune mediated diseases when compared with those of non-immune mediated diseases. The sensitivity and specificity of C3c as a diagnostic test for immune mediated neurological diseases were 61.4 and 100% respectively with a positive and negative predictive value of 100 and 41%. the C3c levels in plasma correlated well with disease severity in MG and GBS patients. Such a correlation was also evident in all CIDP patients except one that had persistent elevation in the presence of clinical improvement. Results suggest that the plasma C3c level may be useful for differentiating immune from non-immune mediated neurological diseases. Plasma C3c may also be used for monitoring disease severity, particularly in myasthenia gravis.

Plasma C3c changes in myasthenia gravis patients receiving high-dose intravenous immunoglobulin during crisis.

Fast-migrating C3c, a sensitive index of complement activation, was assayed in the plasma of 2 myasthenia gravis (MG) patients in crisis who received high-dose IV immunoglobulin therapy. Dramatic responses were observed in both patients. Clinical improvement paralleled a decrement in C3c levels, suggesting that regulation of complement activation may be one possible mechanism of IV immunoglobulin treatment in MG.