High performance liquid chromatographic assay for the determination of Protease Inhibitors (PIs) and Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs) in human plasma.

Objective: To develop and validate a high performance liquid chromatography (HPLC) method for simultaneous quantitative determination of five HIV protease inhibitors (PIs): indinavir (IDV), lopinavir (LPV), nelfinavir (NFV), ritonavir (RTV), saquinavir (SQV), and two non-nucleoside reverse transcriptase inhibitors (NNRTIs): nevirapine (NVP), and efavirenz (EFV) in human plasma. Methods: A sample of 200 µL of plasma and an internal standard were extracted with tert-butyl methyl ether. The compounds were separated on a reversed-phase C18 column with gradient phase of 25 mM phosphate buffer (pH 4.9) and acetonitrile. The limit of quantation, accuracy, precision, specificity, stability and recovery were tested. Results: The lower limit of quantitation for all drugs was 75 ng/mL. The standard curve was linear in the range of 75 ng/mL to 20,000 ng/mL. Intra-day and inter-day variability ranged from 0.1% to 2.4% and 0.3% to 4.1%, respectively. Accuracy ranged from 98.4%-102.4% for three quality controls (75, 100, and 1,000 ng/mL) for all drugs measured. The extraction recovery ranged from 98.7%-101.3%. Conclusion: This method provides a simple, accurate, and precise method for monitoring of plasma concentrations of five PIs and two NNRTIs in the case of weak economy and out of date instrumental limitations.

Guidelines for antiretroviral therapy in HIV-1 infected adults and adolescents: the recommendations of the Thai AIDS Society (TAS) 2008.

BACKGROUND: More than 100,000 patients have been treated, since the implementation of the National Universal Coverage for antiretroviral therapy (ART) in Thailand Although there are several comprehensive guidelines available internationally, there is a need to have guidelines that can be implemented in Thailand. MATERIAL AND METHOD: The guidelines were developed by a panel of 17 members who are the experts on HIV research and/or HIV patient care and appointed without incentive by the Thai AIDS Society (TAS). The recommendations were based on evidences from the published studies and availability of antiretroviral agents. Published studies that are relevant and applicable to Thailand in particular have been taken into consideration. RESULTS: The recommendations include: when to start ART; what to start; how to monitor the therapy; adverse effects and its management; diagnosis of treatment failure; and antiretroviral treatment options in patients with treatment failure. ART in special circumstances, i.e., patients with co-infection of tuberculosis or hepatitis B virus, is also included Appropriate level of CD4+ T-cell count to start ART among Thai patients has been considered carefully. The authors recommend to start ART at CD4+ T-cell count < 200 cells/mm3. CONCLUSION: ART should be initiated in adults and adolescents HIV-1 infected patients with a history of HIV-related illness or AIDS or with a CD4+ T-cell count <200 cells/mm3. For treatment-naive patients, the preferred initial therapy is a non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimen. CD4' T-cell count and viral load should be monitored for at least twice and once a year, respectively. Proper management of antiretroviral-related toxicity and enhancement of adherence are crucial for the long-term success of ART.

Rapid determination of nevirapine in human plasma by high- performance liquid chromatography.

Objective: To develop and validate a high-performance liquid chromatography (HPLC) method for the determination of nevirapine in human plasma. Methods: A plasma sample and an internal standard were extracted with tert.-butyl methyl ether and determined nevirapine concentration by HPLC method. The limit of quantitation (LOQ), accuracy, precision, specificity, stability and recovery were tested for method validation. Results: Standard curve was linear in the range 0.1 ตg/mL to 20 ตg/mL. The limit of quantitation was 0.1 ตg/ml. Coefficients of variation (CV) of intraday and inter-day precision were less than 4 %. Accuracy was range from 97-101%. The extraction recovery was range from 94-112 %. Conclusion: A rapid, sensitive and specific HPLC method was developed and can be used for determination of plasma nevirapine concentration in adult and pediatric patients infected with HIV.

Prevalence of rheumatic disease in HIV infected Thai patients.

OBJECTIVE: To determine the prevalence of rheumatic diseases in HIV-infected patients at Siriraj Hospital, Thailand. MATERIAL AND METHOD: 178 patients who attended the HIV-Clinic at Siriraj Hospital between November 2002 and February 2003 were examined for the presence of rheumatic diseases. Diagnosis of HIV infection was performed by ELISA and confirmed by partial agglutination testing. HIV-infected patients were classified according to the Centers for Disease Control (CDC) 1993 revised classification system. Standard criteria were used to classify the rheumatic diseases. RESULTS: 98 patients had rheumatic diseases. Seventy-seven patients were treated with antiretroviral drugs. Forty-nine patients had mechanical low back pain, twenty-four patients had arthralgia, nineteen patients had plantar fasciitis, eighteen patients had nonspecific myalgia, thirteen patients had fibromyalgia, and eleven patients had others. Arthralgia was associated significantly with Quadricept muscle wasting (p = 0.00001). Nonspecific myalgia was more likely to be associated with female (p = 0. 018) and less likely with use of antiretroviral therapy (p = 0.031). CONCLUSION: Rheumatic diseases were commonly found in HIV-infected patients. Arthralgia associated with wasting Quadricep muscle. Nonspecific myalgia was predominant in female and without antiretroviral drug treatment.

Estimated economic losses of hospitalized AIDS patients at Siriraj Hospital from January 2003 to December 2003: time for aggressive voluntary counseling and HIV testing.

This retrospective study was performed to explore the pattern of adult HIV-infected patients admitted to Siriraj Hospital from January 2003 to December 2003 and estimated the economic losses of these patients. Two hundred and forty four medical records were available for review. The proportion of male to female was 2 to 1. Mean age of patients was 36.64 +/- 9.72 years. The mean CD4 count among 112 patients was 82.79 +/- 96.49 cell/mm3. One hundred and twenty four (50.82%) were newly diagnosed of HIV infection. The three most common opportunistic infections were Tuberculosis (42.62%), Pneumocystis carinii pneumonia (14.75%), and cryptococcosis (13.11%). The mean duration of admission was 15.72 +/- 15.11 days. The mean expense per admission was 38,194.58 +/- 32,354.86 Baht. Fifty four patients (22.13%) died during admission. The mean income of these patients was 3,903.5 +/- 3,841.42 baht per month. The estimated economic losses of 54 patients who died during admission including medical care expense and income losses due to premature death was 69,769,739.32 baht. However, the expected medical expense of antiretroviral medications in these 54 patients if they had been diagnosed earlier and their lives had been saved would have been 42,214,608 baht. Therefore, vigorous voluntary counseling and HIV testing in patients aged 13-70 years when they have any risk factors for HIV infection regardless of symptoms might be more cost effective than diagnosis when they get sick.

Vaccination against hepatitis B virus: are Thai medical students sufficiently protected?

Medical students are frequently at risk of being infected by hepatitis B virus (HBV) via occupational exposure to infected blood or body fluids. In 2002, the Faculty of Medicine, Siriraj Hospital provided screening tests for HBV serology to all medical students for a vaccination campaign against the infection. There were 1,165 medical students tested. Eight hundred and eleven (69.6%) students had immunity by previous vaccination, but more importantly 212 (18.2%) had no immunity and required vaccination. Most of the students who needed to be vaccinated were in the pre-clinical year (82.5%). Moreover, the students in the pre-clinical year who had previous vaccination had a 2.2 times greater risk of having negative anti-HBs than the students in the clinical year (OR = 2.2, 95% CI = 1.4-3.5). This is because they might have been vaccinated when they were young and the antibody waned overtime.

Safety and efficacy of a simplified fixed-dose combination of stavudine, lamivudine and nevirapine (GPO-VIR) for the treatment of advanced HIV-infected patients: a 24-week study.

OBJECTIVE: To determine the efficacy and safety of the fixed-dose combination of stavudine (d4T), lamivudine (3TC) and nevirapine (NVP) in the treatment of antiretroviral naive HIV-infected Thai adults. PATIENTS AND METHOD: An open-label, single arm trial was conducted Baseline clinical assessment and blood test was done on 10, antiretroviral naive HIV-infected patients, who then received a fixed dose combination of d4T, 3TC and NVP (GPO- VIR, Thai Government Pharmaceutical Organization, Bangkok, Thailand). Nevirapine was given as 200 mg once daily for the first 2 weeks. The patients were followed up at 2, 4, 8, 12 and 24 weeks. A CD4 cell count and HIV-RNA assay were done at 12 and 24 weeks. RESULTS: One hundred and one patients were enrolled The mean baseline CD4 cell count and mean HIV RNA were 58.7 (57.7) cells/mm3 and 5.3 (0.5) log10, copies/mL respectively. At week 24th, the mean decrease in log HIV RNA was 3.6 (0.7) log10 copies/mL [P < 0.001; 95% confidence interval (CI), 2.70-3.03]. Eighty one (80.2%) patients had HIV RNA < 400 copies/mL by intention-to-treat analysis (ITT) and 97.6% had HIV RNA < 400 copies/mL by on-treatment analysis (OT). Sixteen (84.2%) patients with baseline HIV RNA < or = 100,000 copies/mL and 65 (82.3%) patients with baseline HIV RNA > 100,000 copies/mL had viral load < 400 copies/mL by ITT (P = 0.842; 95% CI, -20.9%-16.2%). Sixteen (94.1%) patients with baseline HIV RNA < or = 100,000 copies/mL and 65 (98.5%) patients with baseline HIV RNA > 100,000 copies/mL had viral load < 400 copies/mL by OT (P = 0.295; 95% CI, -25.5%-3.8%). The mean CD4 cell count at week 24 was 155.1 (89.0) cells/mm3 (range 13-402). The mean increase in CD4 cell count from baseline was 96.5 (63.5) cells/ mm3 (P < 0.001). A total of 12% of the patients receiving d4T + 3TC + NVP developed skin rashes. Grade 3 or 4 hepatotoxicity was recognized in 7% of the patients. CONCLUSION: Fixed-dose combination of d4T + 3 TC + NVP (GPO- VIR) is safe, well tolerated and effective in increasing CD4 cell counts and suppression of HIV RNA at 24 weeks in advanced HIV-infected patients in Thailand.

Serological survey of viral hepatitis A, B, and C at Thai Central Region and Bangkok: a population base study.

Hepatitis A, B, and C are important viral hepatitis infections in the Thai population. Hepatitis B vaccination was included in the Thai Expanded Program on Immunization (EPI) 10 years ago. In addition, the seroprevalence of hepatitis A has significantly changed in the last two decades. This study was done to evaluate current risk groups for hepatitis A and B infections and identify the magnitude of hepatitis C infection in the general population of Bangkok and six provinces in the Central Region of Thailand, during the period October 2000 to January 2002. This study revealed that the prevalence of anti-HAV in people younger than 25 years was low but very high in people older than 25 years. The prevalence of anti-HAV was 1.95% in Bangkok and 12.7% in other provinces in people younger than 25 years (p<0.001) while 90.9% in Bangkok and 88.2% in other provinces among people older than 25 years. Therefore, people who are older than 25 years should have a blood test for anti-HAV before getting a hepatitis A vaccination. Approximately 80% of people who are not covered by hepatitis B vaccination from EPI are at risk of hepatitis B infection and its complications. This group of people should receive hepatitis B vaccination. For hepatitis C, the prevalence is lower than 2% across age groups and areas. Therefore, current good primary prevention via blood donor screening and health education must be maintained.

HIV infection/acquired immunodeficiency syndrome at Siriraj Hospital, 2002: time for secondary prevention.

The authors retrospectively reviewed the medical records of HIV/AIDS patients who were admitted to the medical service, Siriraj Hospital from January 1, 2002 through December 31, 2002. Demographics, CD4 lymphocyte counts, discharge diagnoses, the incidence of Pneumocystis carinii pneumonia (PCP), cerebral toxoplasmosis and cryptococcosis in patients who received and did not receive appropriate chemoprophylaxis against those opportunistic infections when indicated, and outcome of the patients were collected. Three hundred medical records of 286 HIV/AIDS patients were available for review. One hundred and seventy two patients (60.1%) were male. Mean age of the patients was 36.8 +/- 9.91 years (range 14-74). The mean CD4 lymphocyte count that was determined in 165 patients was 74.7 +/- 134.21 cells/mm3 (range 0-894). Of the 300 admissions, 36 per cent were newly diagnosed HIV infection. Only 23 (7.7%) patients had received antiretroviral drugs at the time of hospitalization. The leading HIV-related diseases were tuberculosis (29.3%), Pneumocystis carinii pneumonia (18.7%), and cryptococcosis (15.7%). The rest of them included cytomegalovirus diseases (6.3%), lymphoma (6.3%), Salmonella bacteremia (6%), cerebral toxoplasmosis (5.7%), cryptosporidiosis (5.3%), disseminated Mycobacterium avium complex infection (1.0%), extrapulmonary histoplasmosis (1.0%), Candida esophagitis (1.0%), progressive multifocal leukoencephalopathy (1.0%), and rhodococcosis (0.7%). Among those for whom HIV infection was established and chemoprophylaxis for PCP, cerebral toxoplasmosis and cryptococcosis were indicated, 9.8 per cent vs 28.2 per cent, 3.6 per cent vs 5.1 per cent, and 10 per cent vs 15.2 per cent of whom received and did not receive the appropriate chemoprophylaxis developed PCP, cerebral toxoplasmosis and cryptococcosis respectively. One hundred and ninety (63.3%) patients were alive at discharge, 84 (28.0%) had died, 21 (7%) were referred to other hospitals, and 5 (1.7%) left hospital against medical advice. The mortality rate in newly diagnosed HIV and in known HIV without antiretroviral treatment were comparable but much lower in known HIV-infected patients who received antiretroviral therapy. Secondary prevention by detection of HIV-infected patients while they are asymptomatic and providing them with appropriate chemoprophylaxis against specific opportunistic infections as well as appropriate antiretroviral treatment would decrease morbidity, mortality, and improve the quality of life of HIV-infected patients in Thailand.

Intracellular production of type I and type II cytokines during HIV-1 progression in Thai patients.

A type I to type II cytokine switch on cells of the immune system has been suggested as a critical step in the etiology of HIV infection. In this study, type I and type II cytokine production of both CD4+ and CD8+ T cells activated by superantigen were investigated in 10 healthy donors and 39 HIV-1 infected patients. Patients were divided into 3 groups based on their CD4 count (< 200, 200-500, > 500 cells/microl). Whole blood from each subject was activated by staphylococcal enterotoxin B (SEB) and anti-CD28. Intracellular cytokine stainings for proinflamatory cytokine (TNF-alpha), type I cytokines (IFN-gamma and IL-2) and type II cytokines (IL-4 and IL-5) in CD4+ and CD8+ T lymphocytes were determined by flow cytometer. Type I cytokine (IFN-gamma) expression in CD4+ T cells co-expressing with CD69 were significantly increased in HIV infected patients, particularly in patients with CD4 counts < 200 and 200-500 cells/microl (means +/- S.D. of 20.7 +/- 18.7% and 10.5 +/- 5.9%, respectively) when compared with 4.8 +/- 1.8% in the normal group (p < 0.05). But IL-2 production in both groups of patients was significantly lower than the normal (3.8 +/- 2.6% and 3.2 +/- 1.4% in patients with < 200, 200-500 cells/microl, and 5.9 +/- 1.5% in the normal group) (p < 0.05). For type II cytokines, there was no difference in all groups of subjects when IL-4 was determined. However, IL-5 production was significantly higher in patients with a CD4 count < 200 cells/microl (0.6 +/- 0.5%) than that in the normal group (0.1 +/- 0.1%) (p < 0.005). CD8+ T cells also showed higher IFN-gamma production in patients with a CD4 count < 200 cells/microl (11.9 +/- 4.7%) and 200-500 cells/microl (12.0 +/- 4.3%) than the normal group (5.3 +/- 2.5%) (p < 0.005). In contrast, IL-2 production in CD8+ T cells was low in these HIV infected patients (0.3 +/- 0.2%, 0.3 +/- 0.2%, and 0.3 +/- 0.4% in patients with < 200, 200-500, and > 500 cells/microl, respectively), which was significantly different compared to the control group (1.2 +/- 0.8%) (p < 0.05). For type II cytokines, only IL-4 production in patients with a CD4 count < 200 cells/microl (0.1 +/- 0.1%) was significantly reduced when compared to the other groups (p < 0.05). This study shows that although HIV infection alters the production of both type I and type II cytokines, it does not induce a polarized type I or type II state in the course of HIV-1 progression in Thai patients.

Infection due to nontuberculous Mycobacterium other than MAC in AIDS patients at Siriraj hospital during 1998-2000: saprophyte vs pathogen.

HIV is a major health problem in Thailand. These patients are vulnerable to opportunistic infections, especially Mycobacterium tuberculosis and MAC infection. However, NTM was considered a rare disease in Thailand before the AIDS era. In this study, there were 38 HIV seropositive patients with NTM (other than MAC) identified from clinical specimens during the 3 year period 1998-2000 at Siriraj Hospital, which has a higher prevalence than the previous report. Among these patients, 29 cases were likely to have had definite infection from NTM, 5 cases possibly had NTM as a pathogen, and 4 cases had NTM as colonization. The most common site of infection was the lung (87%) and most common symptoms were cough (62.2%), fever (34.2%), weight loss (42.1%), and lymphadenopathy (5.3%). The outcome was poor because many NTM are not susceptible to standard medication for tuberculosis which is the empirical treatment for the majority of HIV seropositive patients with a clinical finding suspected of mycobacterial infection. The fatality rate was as high as 58.6 per cent. Awareness of NTM as a potential pathogen in HIV seropositive patientsand adjustment of medications even before the availability of culture results may improve the outcome of treatment of NTM infection in HIV seropositive patients.

Occupational exposure to HIV among healthcare workers: Experience at Siriraj Hospital.

Objective : To evaluate the incidence and epidemiological data concerning occupational exposure to HIV among healthcare workers (HCWs) at Siriraj Hospital. Methods : We prospectively collected data concerning occupational exposure among HCWs at Siriraj Hospital using a self-reporting system from 1994 to 2001. All exposed individuals were followed up for at least 6 months after exposure. Results : During the years 1994-2001, there were 691 incident reports from 670 individuals, 180 males and 490 females. The groups of HCWs exposed to blood and other body fluids consisted of : - 152 (22.0%) nurses, 147 (21.3%) medical students, and 119 (17.2%) externs. Needlestick or sharps injuries accounted for 538 (77.9%) cases, while the number exposed to mucous membranes was 93 (13.5%). The remaining 28 (4.1%) cases were of broken skin and 32 (4.6%) cases of intact skin exposure. Venepuncture was the procedure with highest risk of producing injury (20.7%). The incidents mostly occurred on medical wards 156 (22.6%) cases, operating rooms 121 (17.5%) cases, and in the emergency room 111 (16.1%) cases. The HIV status of the sources was positive in 230 (33.3%) cases, negative in 315 (45.6%) cases, and unknown in 146 (21.1%) cases. Postexposure prophylaxis was given in 206 (29.8%) cases. In terms of HIV seroconversion, only 172 (27.3%) out of 629 cases reported back negative results for anti-HIV at the 6 month follow-up. Conclusion : Occupational exposure to HIV at Siriraj Hospital occurred most frequently among nurses, medical students, and externs as needlestick or sharps injuries on medical wards. According to our data up to this time, no one seroconverted or claimed compensation for work-related HIV infection.

Multidrug resistant mycobacterium tuberculosis infections in patients with acquired immunodeficiency syndrome at Siriraj Hospital.

Bachground : The increase in the prevalence of multidrug-resistant tuberculosis (MDR-TB) has been alarming globally. HIV and Mycobacterium tuberculosis co-infection is not uncommon. The mortality rate of MDR-TB it self is generally high. We describe 21 patients with multidrug-resistant Mycobacterium tuberculosis infection (MDR-TB), who had acquired immunodeficiency syndrome (AIDS). Methods: Information concerning all patients with AIDS and M. tuberculosis co-infection treated at Siriraj Hospital was collected from January 1, 1998, through June 30, 2001 which included clinical presentation, laboratory data, chest x-ray findings, treatment and outcome. Results: Six hundred and forty nine isolates of M. tuberculosis were tested for antimycobacterial susceptibility. 58 isolates (8.9%) were multidrug-resistant. Twenty one patients (36%) had AIDS. Of these, seven patients (33.3%) had primary drug resistant tuberculosis and 15 (66.7%) had secondary drug resistant tuberculosis. The mortality rate of MDR-TB with AIDS was 71.4%. The most common clinical presentations were fever (61.9%), cough (47.6%), and peripheral lymphadenopathy (33.3%). Twenty four percent had pulmonary disease only, 52% had both pulmonary and extrapulmonary diseases. The mean CD4 lymphocyte count was 38.8+45.77 cells/?L. Thirty three percent of the isolates were resistant to isoniazid and rifampicin only, 43% were resistant to 3 drugs and 24% to 4 drugs. Conclusion: The prevalence of MDR-TB was 8.9% ( 58 of 649 cases) in Siriraj Hospital from January 1, 1998 through June 30, 2001. Twenty-one of these patients (36%) had AIDS. Patients with MDR-TB were more likely to have disseminated disease and low CD4 lymphocyte counts. The mortality rate was high. All isolates of M. tuberculosis should be tested for antimycobacterial susceptibility to guide treatment and improve the outcome.

Leptospirosis 2001.

A 20-year-old man was admitted to Siriraj Hospital because of high fever and jaundice. He also had acute renal failure. According to the other clinical manifestations and laboratory investigations, the differential diagnosis included severe systemic infections, especially leptospirosis and scrub typhus. A definitive diagnosis was obtained by a positive microscopic agglutination test for Leptospira interrogans, serovar bratislava. Serological tests for scrub typhus and dengue infection were negative. The number of patients with leptospirosis has been increasing in many hospitals and outbreaks of the disease have been reported in northeastern Thailand since 1997. In October 1999, Leptospira interrogans, serovar pyrogenes was isolated from the blood of a febrile patient with clinical leptospirosis in Burium province. This pathogen was used to study the progressive microanatomical changes within organs including the kidney, lungs, liver, gastrocnemius and hamstring muscles of infected hamsters. The kidney showed degenerative changes of the renal tubular cells and many pathological appearances of the glomerular tuft. Interstitial nephritis and pyelonephritis were also found. In the lungs, the alveolar and interalveolar capillaries were engorged with red blood cells. Both bronchopneumonitis and interstitial pneumonitis were observed. The liver showed cloudy swelling of hepatocytes which lead to dissociation of the hepatic cords. Vascular and sinusoical congestion, prominent Kupffer cells and inflammatory cell infiltration in the parenchyma, and sinusoids as well as the portal area were demonstrated. Hepatocellular necrosis was found scattered throughout the hepatic lobules. Some hamsters showed blood vessel congestion in the gastrocnemius and hamstring muscles. Inflammatory cell infiltration was shown in the perimysium of the gastrocnemius muscle of one hamster. Another showed necrosis of some muscle fibers together with inflammatory cell infitration which are sings of muscular inflammation. The prevention and control of Leptospirosis is discussed.