Estimation of Malaria Parasite Densities by Different Formulas in Thailand

Introduction: Although there are many methods in malaria diagnoses e.g., quatitative buffy coat (QBC), rapid diagnosis tests (RDTs), serological tests and molecular diagnosis methods such as PCR, but microscopy still remains the gold standard for malaria diagnosis. Estimation of malaria parasite density can be carried out by using assumed white blood cells (WBC) and red blood cells (RBC) counts. Objective: The aims of this study were to determine malaria parasite densities calculated by Original Research Article assumed WBC and RBC counts; and to compare their reliability with absolute WBC and RBC counts. Methods: The clinical presentations and laboratoryfindings of specimens collectedfrom 512 uncomplicated falciparum and vivax malaria patients admitted to Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand were utilized and analysed for estimation of malaria parasite densities by using different formulas.Results: Parasite densities calculated by WHO recommended assumed WBC of 8,000 /μL, and assumed RBC counts of 4.7x106-6.1x106/μL and 4.2x106-5.4x106 /μL for males and females respectively led to overestimation, and resulted in low reliability when compared to the absolute WBC and RBC counts.Parasite densities calculated by assumed WBC of 5,900/μL in thick blood; by assumed RBC of 4.8x106/μL for malesand 4.3x106/μLfor femalesin thin blood film respectively gave more precise estimation.Conclusion: Assumed WBC and RBC counts for calculating malaria parasite densities haveto be adjusted to use in Thailand for more precise estimation. Parasite densities calculated by assumed WBC and RBC used in other malaria endemic countries might need further re-evaluation

Efficacy of Artequick versus artesunate-mefloquine in the treatment of acute uncomplicated falciparum malaria in Thailand.

To determine the efficacy, safety and tolerability of an alternative short-course, artemisinin-based combination therapy for acute uncomplicated Plasmodium falciparum malaria, we compared Artequick--a fixed-dosed combination of artemisinin (80 mg), piperaquine (400 mg), and primaquine (4 mg), per tablet--with a standard regimen of artesunate-mefloquine. A total of 130 patients were randomly assigned to treatment with an orally administered, once-daily, 3-day regimen of either Artequick (Group A: 3.2 mg/Kg/day of artemisinin, 16 mg/Kg/day of piperaquine, and 0.16 mg/Kg/day of primaquine) or artesunate-mefloquine (Group B: artesunate, 4 mg/Kg/day, with mefloquine, 8 mg/Kg/day). Patients receiving each regimen had a rapid clinical and parasitological response. All treatments were well tolerated, and no serious adverse effects occurred. No significant differences were found in fever- and parasite-clearance times between the two study groups. The 28-day cure rates were similarly high, at 98.5% and 100%, in groups A and B, respectively. We conclude that Artequick was as effective and well tolerated as artesunate-mefloquine and could be used as an alternative treatment for multidrug-resistant Plasmodium falciparum malaria in Southeast Asia.

Dose ranging studies of new artemisinin-piperaquine fixed combinations compared to standard regimens of artemisisnin combination therapies for acute uncomplicated falciparum malaria.

To determine the optimum dose of artemisinin-piperaquine combination therapies for acute uncomplicated Plasmodium falciparum malaria, we examined 7 candidate regimens in 411 patients admitted to the Bangkok Hospital for Tropical Diseases. The studies were performed from May 2005 to October 2005 and November 2005 to June 2006. We compared 3-day courses of artesunate-mefloquine, artemether-lumefantrine (Coartem) and of dihydroartemisinin-piperaquine (Artekin) as reference antimalarial treatments, with candidate regimens using 2-3 day courses of artemisinin-piperaquine, Artequick. Initially, patients receiving each of the regimens had a rapid clinical and parasitological response. All treatments were well tolerated and no serious adverse effects occurred. The 28-day cure rates were < 80% for the 2-day treatments with artemisinin-piperaquine at 2.4 mg/kg and 14.4 mg/kg, respectively, in the first study period and artemisinin-piperaquine at 3.2 mg/kg and 16.0 mg/kg, respectively, but > 98% for the 3-day regimens. These results suggest that a 3-day course of artemisinin-piperaquine at 3.2 mg/kg and 16.0 mg/kg, respectively, deserve further evaluation as an alternative treatment for multidrug-resistant P. falciparum malaria.

A quantitative ultrastructural study of the liver and the spleen in fatal falciparum malaria.

We performed a retrospective study of 25 patients who died of severe falciparum malaria in Thailand and Vietnam using electron microscopy. The aims of the study were: to determine if there was any significant association between parasitized red blood cells (PRBC) sequestered in liver and spleen and particular pre-mortem clinical complications, and to compare the degree of parasite load between the liver and spleen within the same patients. PRBC sequestrations in each organ were compared with the pre-mortem parasitemia, to calculate the sequestration index (S.I.). The S.I. showed that the degree of PRBC sequestration in the spleen was higher than the liver (S.I. median = 3.13, 0.87, respectively) (p < 0.05). The results of quantitative ultrastructural study showed a significantly high parasite load in the liver of patients with jaundice, hepatomegaly and liver enzyme elevation (p < 0.05). We found a significant correlation between PRBC sequestration in the liver and a high serum bilirubin level, a high aspartate aminotransferase (AST) level and an increase in the size of the liver (Spearman's correlation coefficient = 0.688, 0.572, 0.736, respectively). Furthermore, a higher parasite load was found in the liver of patients with acute renal failure (ARF) compared to patients without ARF (p < 0.05). These findings suggest that PRBC sequestration in the liver is quantitatively associated with pre-mortem hepatic dysfunction and renal impairment. There was no significant difference between splenomegaly and PRBC sequestration. The size of a palpable spleen was not correlated with parasite load in the spleen. When ultrastructural features were compared between the two reticuloendothelial organs, we found that the spleen had more PRBC and phagocytes than the liver. The spleen of non-cerebral malaria (NCM) patients had more phagocytes than cerebral malaria (CM) patients. This observation reveals that the spleen plays a major role in malaria parasite clearance, and is associated with host defence mechanisms against malaria.

Hematopoietic features and apoptosis in the bone marrow of severe Plasmodium falciparum-infected patients: preliminary study.

The mechanism of anemia in severe falciparum malaria is still not completely understood. The purpose of this study was to determine whether apoptosis in the erythroid lineage causes anemia in falciparum malaria. Bone marrow aspirated from 8 severe falciparum malaria patients, 3 normal volunteers and 5 retrospective normal bone marrow smears were investigated. By light microscopic study, 5 of 8 hyperparasitemic patients had hypocellular bone marrows and erythroid hypoplasia, whereas the other 3 patients had normal cellularity. The mean myeloid : erythroid ratio of these 5 patients was significantly (p < or = 0.05) higher than normal. Apoptosis of bone marrow nucleated cells (BMNC) could be determined from the exposure of phosphatidylserine (PS) on the cell membrane but not DNA fragmentation (180-250 bp) or ultrastructural morphology. The percentages of apoptotic BMNC and apoptotic erythroid cells in bone marrow from each patient and controls varied from low to high, and were not associated with parasitemia. This study suggests that destruction of erythroid lineage, particularly through apoptosis regulation, cannot solely account for anemia in falciparum malaria.

The efficacy and adverse effects of GPO-VIR (stavudine+lamivudine+nevirapine) in treatment-naïve adult HIV patients.

A descriptive, combined retrospective and prospective study was conducted at the Anonymous Clinic, Chon Buri Hospital, Chon Buri Province, Thailand from November 10, 2003 to January 4, 2004. A total of 83 adult HIV-treatment-naïve patients undergoing treatment with GPO-VIR (stavudine, lamivudine, and nevirapine) for at least one year were studied. The objectives of the study were to assess the efficacy of GPO-VIR by evaluating body weight changes, CD4 T-cell count changes, the occurrence of opportunistic infections, and long-term side effects, such as lipodystrophy, during treatment. Of 83 studied patients, approximately half (52.3%) of them had a body weight increase > 10% of pre-treatment body weight after 12 months treatment. After taking GPO-VIR, CD4 T-cell counts increased rapidly, by a median of 78 x 10(6) cells/l during the first three months. 39.5% of the patients attained median CD4 counts > 200 x 10(6) cells/I, and 11.6% achieved > 500 x 10(6) cells/l after 2 years of treatment. The occurrence of opportunistic infections was significantly lower after treatment with GPO-VIR (p = 0.001). Subjective assessment of lipodystrophy by physicians and patients showed that 16.8% had symptoms of lipodystrophy within 2 years of GPO-VIR treatment. There was a significant association between older age group (40-49 years) and occurrence of lipodystrophy (p = 0.043). GPO-VIR is an inexpensive and effective antiretroviral drug regimen for initiating treatment of naïve patients, but careful assessment for lipodystrophy is necessary, especially after one year of treatment.

The prevalence of nonalcoholic steatohepatitis in Thai patients with non-HBV, non-HCV chronic hepatitis.

This study aimed to determine the prevalence of nonalcoholic steatohepatitis in Thai patients with non-HBV, non-HCVchronic hepatitis. The clinical and laboratory findings associated with non alcoholic steatohepatitis were discussed. Forty-six patients with negative markers for viral hepatitis B and viral hepatitis C and no history of alcohol consumption or consumption less than 20 grams of ethanol per day were recruited. The informed consent for liver biopsy and blood collecting to identify the etiology of chronic hepatitis was performed. Most patients (76.1%) exhibited fatty metamorphosis of the liver which included steatosis (21.8%) as well as steatohepatitis (54.3%). Eleven of 46 patients (23.9%) were classified as cryptogenic chronic hepatitis. There were statistically significant differences between the fatty metamorphosis group and the cryptogenic chronic hepatitis group with regard to the fasting blood sugar, serum alkaline phosphatase, serum ferritin and histologically necroinflammatory grading score (p < 0.05). Between the steatosis group and the steatohe-patitis group, body mass index (BMI) was the only factor showing statistically significant difference (p = 0.02). Eight from 25 NASH-patients had diabetes mellitus (32.0%) and the AST to ALT ratio in this group was 0.6. The histopathological assessment for inflammation and fibrosis by using Knodell score, the fibrosis score which equal or higher than 3 was found in 20.0% of NASH-patients. CONCLUSION: The prevalence of NASH-patients in Thai patients, with non HBV, non HCV chronic hepatitis was 76.1%, while the liver biopsy can add the diagnostic yield especially in the group of unexplained chronic hepatitis with obesity, diabetes mellitus and dyslipidemia.

Declining mefloquine sensitivity of Plasmodium falciparum along the Thai-Myanmar border.

Mefloquine sensitivity of Plasmodium falciparum along the Thai-Myanmar border, both in vitro and in vivo, following different first-line treatments for uncomplicated falciparum malaria patients in these areas during the period 1997--2003 were studied. Standard in vitro micro tests and in vivo efficacy according to World Health Organization methodologies were performed. P. falciparum isolates along the Thai-Myanmar border with in vitro sensitivity to mefloquine have had up to a ten-fold decrease in sensitivity compared to a baseline done in 1986, conducted one year after the drug was first introduced to Thailand. The reduction in the mefloquine sensitivity of P. falciparum isolates in Tak Province developed rapidly, with the highest IC50 of 1,254 nM in 1997. The IC50 declined to 1,067 and 737 nM in 1999 and 2001, respectively, but there was no statistically significant difference in the sensitivity. The sensitivity of P. falciparum isolates from Mae Hong Son, Kanchanaburi, and Ranong, where the first line treatment was mefloquine 15 mg/kg single dose, continued to decline, where in 2001 the IC50 were 1,087, 941, and 1,116 nM, respectively, in these provinces. The difference in sensitivities of P. falciparum isolates in Mae Hong Son and Ranong in 2001, compared to 1997, was statistically significant (p<0.05). Good therapeutic efficacy of the artesunate-mefloquine combination in Tak Province was observed. Adequate clinical responses (ACR) were 89.5% and 92.3% in 1997 and 2002, respectively. The efficacy of mefloquine alone in Mae Hong Son, Kanchanaburi, and Ranong has significantly dropped. ACR in 1997 and 2001 in Mae Hong Son were 87.8% and 73.2%, respectively, in Kanchanaburi were 82% and 59.6%, respectively, and in Ranong were 96% and 31.6%, respectively.

Association of intestinal helminths with decreased liver size and sCD23 concentration during falciparum malaria.

To determine if intestinal helminths and the CD23/nitric oxide pathway had an influence on liver size, we conducted a cross-sectional study on 438 patients with confirmed P. falciparum malaria admitted at the Hospital for Tropical Diseases in Bangkok. For all patients the liver size was measured as number of centimeters below the rib cage, a stool examination was conducted, and CD23 and reactive nitrogen intermediates were measured. The median liver size was smaller in helminth-infected patients than in helminth-free patients (chi2 for trend = 9.1, p = 0.003). Liver size significantly increased with the concentration of sCD23 (p < 0.0001). The median sCD23 concentration (OD) was significantly lower in helminth-infected patients than in helminth-free patients, respectively 0.33 (quartiles 0.24-0.57) and 0.45 (quartiles 0.27-0.59), (p = 0.01). There was a negative correlation between sCD23 concentrations and RNI (Spearman's rho = -0.40, p < 0.0001). All the above results remained significant after controlling for potential confounders. These results are compatible with a CD23/NO-mediated decrease in liver size in helminth-infected patients.

Clinical experience with intravenous quinine, intramuscular artemether and intravenous artesunate for the treatment of severe malaria in Thailand.

We prospectively studied 803 Thai patients admitted to the Bangkok Hospital for Tropical Diseases to assess the safety, tolerability and effectiveness of treatments for strictly defined P. falciparum malaria. Patients were assigned to one of five treatment groups: (i) a 5-day course of intravenous artesunate in a total dose of 600 mg, Group Aiv; (ii) intravenous artesunate as in Group Aiv followed by mefloquine, 25 mg/kg, Group Aiv+M; (iii) a 3-day course of intramuscular artemether in a total dose of 480 mg, Group Aim; (iv) intramuscular artemether as in Group Aim followed by mefloquine, 25 mg/kg, Group Aim+M, and (v) intravenous quinine, 200 mg/kg given in divided doses over seven days followed by oral tetracylcine, 10 mg/kg, for 7 days. When patients could take oral medications, the parenteral antimalarials were administered as oral agents. There were no major adverse effects observed with any of the five treatment regimens. With all regimens, 95 to 100% of the patients survived. Mean parasite clearance times were more rapid with the artemisinin regimens (53 to 62 hours) than with quinine (92 hours). The mean fever clearance times with intravenous artesunate (80 to 82 hours) were about a day shorter than those with intramuscular artemether (108 hours) or intravenous quinine (107 hours). Mefloquine reduced the recrudescence rate from 24 to 5% with intravenous artesunate but from 45 to 20% with intramuscular artemether; recrudescence was 4% with quinine and tetracycline. A dose and duration of therapy greater than those in this study are needed for optimal therapy with intramuscular artemether. Effective therapy for severe falciparum malaria can be provided by either intravenous artesunate followed by mefloquine or by intravenous quinine followed by tetracycline.

Septicemic melioidosis and meningitis: A case report.

Melioidosis predominantly affects the rice-farming community of north-eastern Thailand, and occurs mainly in patients with underlying diseases such as diabetes mellitus or chronic renal impairment. It may be asymptomatic or may present as acute septicaemic or chronic suppurative infection. Neurological involvement in melioidosis is uncommon. We report here on a patient with septicaemic melioidosis and meningitis. A 48-year-old man presented with a three-month history of fever, chillis and weight loss. He came from Udon Thani and was a known diabetic who had been on regular treatment for three years. Both Widal and Weil-Felix reactions were negative with normal X-ray of the chest. On the next day, the patient had severe headache with a stiff neck. The CSF contained elevated glucose and protein concentrations, polymorphonuclear cells and lymphocytes but revealed no growth on culture. Two days later, the patient developed a cough, dyspnoea with difficulty in swallowing. Culture frpm the blood and sputum yielded pure Burkholderia pseudomallei. The patient was treated with a combination of ceftriaxone and trimethoprim-sulphamethoxazle which was found to be sensitive in vitro. However, the patient died on day 7 of hospitalization. This case illustrate an urgent need for early diagnosis and identification of sensitive drugs for the treatment the melioidosis which is still quite common in north. Eastern Thailand.

Serum transcobalamin II level in a malarial patient with ceftriaxone-induced haemolyic anaemia.

Cephalosporins have rarely been reported as the cause of immune haemolytic anaemia (IHA). The case history of a patient who had elevated serum transcobalamin II (TCII) levels due to a ceftriaxone-induced haemloytic anaemia is presented in this study. The patient was admitted because of high fever due to P.falciparum. The fever subsided after treatment with anti-malarial drugs. However, two days later, the fever recurred and ceftriazone was given. On the next day, the patient had haemolysis with haemoglobinuria and renal insufficiency which resolved after withdrawal of the drug. Serum TCII levels were elevated during the haemolytic episode and the period of renal impairment. The mechanisms of increased serum TCII are probably due to the acute haemolysis and nephrotoxicityinduced by ceftriaxone, leading to the impaired catabolism and clearance of TCII. Therefore, intravascular THII survival is prolonged. Resulting in elevated serum TCIIlevels.

Persistently elevated serum transcobalamin II in a patient with reactive haemophagocytic syndrome.

A 24-year-old man was admitted to the hospital with a history of prolonged fever, peripheral blood neutropenia and bone marrow showing benign haemophagocytic histiocytosis. He presented with symptoms and manifestations over a brief duration until death, with the progressive development of multi-organ dysfunction. His serum TCII levels were persistently elevated throughout the disease duration in the hospital. Available evidence indicates that macrophages, mononuclear cells and histiocytes can produce TCII. Serum TCII levels in patients with reactive haemophagocytic syndrome are therefore elevated due to the increased be helpful in making the diagnosis in these patients.

Effect of chloroquine on serum vitamin B12 and its binding proteins in patients with plasmodium vivax.

Chloroquine (CQ) is widely used as an antimalarial agent. It is accumulated in the lysosomes of various types of cells and inhibits the intralysosomal degradation of a wide range of proteins. CQ is found to interfere with vitamin B12 transportation in vitro and in the experimental animal. The present study was performed in order to determine such interference in humans receiving CQ. Serum vitamin B12 and vitamin B12 binding proteins were determined in 13 patients with P.vivax, both before and after receiving six tablets and 10 tablets of CQ. There were no significant differences between serum vitamin B12, UBBC, TBBC and TC values in patients before and after taking 6 tablets of CQ. A slight but not significant decrease in serum vitamin B12 and a significant increase in serum TCII were found after taking 10 tablets of CQ. The low serum vitamin B12 could be due to the effect of CQ on prevention of lysosomal degradation of intrinsic factor, leading to the accumulation of IF-B12 in the intestinal mucosa. As CQ also inhibits the intracellular degradation of TCII, therefore, more TCII levels are synthesized and secreted from various organs by the feedback mechanism. All these findings indicated the CQ had some effects on vitamin B12 absorption and transportation. However, these effects were relatively unsevere for the required dose of CQ in the treatment of P.vivax malaria.

Idiopathic hypereosinophilic syndrome: A case report.

Eosinophilia is quite common in Thailand due to the high prevalence of parasitic diseases. A study in 1993-1994 showed that 41% of patients with different parasitic infections had eosinophilia. We report herein a patient with idiopathic hypereosinophilic syndrome (HES). A 23-year-old man was admitted to the hospital with a 1.5 month history of chest pain and dyspnea on exertion. He had symptoms and signs of congestive heart failure. His peripheral blood revealed eosinophils 42% (8.4 x 109/1) with many hypogranulated and vacuolated eosinophils. Bone marrow aspiration showed a hypercellular marrow with eosinophils 10% and plasma cells 5-10%. Chest x-ray showed cardiomegaly and an echocardiography revealed pericardial effusion. As this patient had eosinophilia, signs and symptoms of cardiac involvement and lack of evidence for parasitic, allergic or other causes of eosinophilia, a diagnosis of idiopathic HES was made. Treatment with prednisolone resulted in the improvement of congestive heart failure and the eosinophil count returned to normal. A prompt diagnosis and treatment are needed in patents with this syndrome.

Tropical pyomyositis: A case report.

Tropical pyomyositis is an inflammatory condition of skeletal muscles, usually associated with infection with microbials. As this disease is quite rare in Thailand, we report here a 20-year-old man, a Burmese immigrant in Bangkok for 5 months, who presented with fever, malaise, luecocytosis, elevated sedimentation rate, anemia and a tender mass on his right back. Ultrasonography revealed a mass with abscess suggested tropical pyomyositis and was confirmed by open surgery. Pus was sterile. After treatment with cloxacillin for 24 days, the abscess healed and the patient was discharged and has been followed for 4 months with no recurrence of pyomyoistis. Tropical pyomyositis should be considered and differentiated from viral infection, osteomyelitis and visceral abscesses in the tropical countries.

Salmonella choleraesuis bacteremia associated with The Acquired Immunodeficiency Syndrome (AIDS).

Multiple opportunistic infections are characteristic of the acquired immunodeficiency syndrome (AIDS). An emerging problem with salmonella infection among patients with AIDS is increasing. We report here 3 patients with AIDS who presented with fever and were found to be bacteremia due to serotypes of Salmonella choleraesuis. As this serotype rarely occurs in normal hosts in Thialand, finding the infection in patients with AIDS indicate that these patients are at high risk. It suggests that AIDS patients with prolonged febrile illness should be investigated for salmonella infection.

Elevated serum transcobalamin II levels in patients with prolonged fever.

Serum transcobalamin II levels were determined in 70 patients with prolonged fever. Twelve patients were found to have elevated serum TCII levels, i.e., 8 patients with salmonellosis, 3 patients with scrub typhus and 1 patient with pyrexia of unknown origin. There were no relationships between serum TCII levels and white blood cells, lymphocytes or monocytes. The possible mechanism producing increased serum TCIII levels in patients with salmonellosis and scrub typhus is the increased synthesis and release of TCII by the proliferative mononuclear phagocytic cells of the reticuloendothlial tissues such as spleen, liver, bone marrow and lymph nodes. This study gives the additional data that elevated serum TCII may occur not only in inflammatory disorders, autoimmune diseases, lymphoproliferative disorders, malignant histiocytosis and neoplasms, but also in infection with salmonellosis and scrub typhus.

Salmonella infection in patients with plasmodium falciparum malaria.

Patients with P.falciparum malaria infection are unusually susceptible to a variety of infectious diseases. There is an increased incidence of salmonella infection and bacteremia in patients with malaria. The objective of the present study was to report 3 patients with P.falciparum malaria infection in association with salmonellosis. These patients presented with fever and their blood smears revealed P.falciparum. They were treated with anti-malarial drugs. The malarial parasites disappeared from the peripheral blood, however, the fever persisted or recurred within a few days. Blood cultures grew S.choleraesusis, S.enteritidis and S.paratyphi A, respectively. These patients were treated successfully with antibiotics. Blood cultures should therefore be performed in all patients with P.falciparum malaria whose fever persists after treatment with antimalarial drugs.