Validation and Clinical Evaluation of a Novel Method To Measure Miltefosine in Leishmaniasis Patients Using Dried Blood Spot Sample Collection.

To facilitate future pharmacokinetic studies of combination treatments against leishmaniasis in remote regions in which the disease is endemic, a simple cheap sampling method is required for miltefosine quantification. The aims of this study were to validate a liquid chromatography-tandem mass spectrometry method to quantify miltefosine in dried blood spot (DBS) samples and to validate its use with Ethiopian patients with visceral leishmaniasis (VL). Since hematocrit (Ht) levels are typically severely decreased in VL patients, returning to normal during treatment, the method was evaluated over a range of clinically relevant Ht values. Miltefosine was extracted from DBS samples using a simple method of pretreatment with methanol, resulting in >97% recovery. The method was validated over a calibration range of 10 to 2,000 ng/ml, and accuracy and precision were within ±11.2% and ≤7.0% (≤19.1% at the lower limit of quantification), respectively. The method was accurate and precise for blood spot volumes between 10 and 30 µl and for Ht levels of 20 to 35%, although a linear effect of Ht levels on miltefosine quantification was observed in the bioanalytical validation. DBS samples were stable for at least 162 days at 37°C. Clinical validation of the method using paired DBS and plasma samples from 16 VL patients showed a median observed DBS/plasma miltefosine concentration ratio of 0.99, with good correlation (Pearson'sr= 0.946). Correcting for patient-specific Ht levels did not further improve the concordance between the sampling methods. This successfully validated method to quantify miltefosine in DBS samples was demonstrated to be a valid and practical alternative to venous blood sampling that can be applied in future miltefosine pharmacokinetic studies with leishmaniasis patients, without Ht correction.

Malnutrition in Healthy Individuals Results in Increased Mixed Cytokine Profiles, Altered Neutrophil Subsets and Function.

Malnutrition is commonly associated with increased infectious disease susceptibility and severity. Whereas malnutrition might enhance the incidence of disease as well as its severity, active infection can in turn exacerbate malnutrition. Therefore, in a malnourished individual suffering from a severe infection, it is not possible to determine the contribution of the pre-existing malnutrition and/or the infection itself to increased disease severity. In the current study we focussed on two groups of malnourished, but otherwise healthy individuals: moderately malnourished (BMI: 18.4-16.5) and severely malnourished (BMI <16.5) and compared several immune parameters with those of individuals with a normal BMI (≥18.5). Our results show a similar haematological profile in all three groups, as well as a similar ratio of CD4+ and CD8+ T cells. We found significant correlations between low BMI and increased levels of T helper (Th) 1 (Interferon (IFN)-γ, (interleukin (IL)-2, IL-12), Th2 (IL-4, IL-5, IL-13), as well as IL-10, IL-33 and tumor necrosis factor-α, but not IL-8 or C reactive protein. The activities of arginase, an enzyme associated with immunosuppression, were similar in plasma, peripheral blood mononuclear cells (PBMC) and neutrophils from all groups and no differences in the expression levels of CD3ζ, a marker of T cell activation, were observed in CD4+ and CD8+T cells. Furthermore, whereas the capacity of neutrophils from the malnourished groups to phagocytose particles was not impaired, their capacity to produce reactive oxygen species was impaired. Finally we evaluated the frequency of a subpopulation of low-density neutrophils and show that they are significantly increased in the malnourished individuals. These differences were more pronounced in the severely malnourished group. In summary, our results show that even in the absence of apparent infections, healthy malnourished individuals display dysfunctional immune responses that might contribute to increased susceptibility and severity to infectious diseases.

Staple Ethiopian diet and cancer of the oesophagus.

BACKGROUND: Previous reports from Ethiopia suggest that oesophageal cancers are due to thermal injury of hot food and beverages. However, in most other countries diet plays an important role. OBJECTIVES: To investigate the association of diet and incidence of carcinoma of oesophagus in Ethiopia. DESIGN: A prospective study. SETTING: All adult patients, who were either referred or self referred from other health institutions, were investigated in Mexico Higher Clinic, Addis Ababa, Ethiopia. SUBJECTS: All patients who presented for endoscopy for the duration of one year, February 1st to January 2002, were included in the study. RESULTS: There were 902 adult patients of whom 596 (66%) were male and 306 (34%) were female. The age range was 16 to 81 with a mean of 37 and the male to female ratio was 1.95:1. Christians out-numbered Muslims. There were more merchants than office workers or peasants. Those who came from Addis Ababa were 600 (66%) and the rural patients were 302 (34%). The ethnic groupings indicated Amhara, Gurage and Oromo to be the predominant ones. Six hundred sixty two patients ate tef, 140 ate wheat and 100 ate kocho (Qocho). There were 27 specimens, which had malignant cells. Thirteen specimens failed to show malignant cells. Thirteen (13%) Qocho eaters had cancer of oesophagus, nine (6.5%) wheat eaters had cancer and only five of tef eaters (0.70%) were affected. Dysphagia was the single most common symptom with which patients presented in cases of cancer of oesophagus. Cancer of oesophagus was located in upper, middle and EG junctional area in 5, 12 and 10 patients respectively. There were 16 cases of squamous cells and 11 cases of adenocarcinoma of a total of 40 specimen taken. Thirteen specimens failed to show malignant cells. CONCLUSION: Kocho (Qocho) eating appearsto be associated with developing cancer of the oesophagus.

Transmission of hepatitis B virus infection in Ethiopia with emphasis on the importance of vertical transmission.

This two-year controlled study was conducted to define the mode of transmission of hepatitis B infection in Ethiopia. Twenty-five of 500 (5%) expectant mothers were identified as HbsAg positive (Group A). A control group of 29 mothers negative for HBsAg (Group B) was randomly selected. These 54 mothers and their families (husbands, infants and the infants' older siblings) were followed for two years with determinations of serological markers of ongoing or past hepatitis B virus infection at 6, 12 and 24 months. Social and clinical data as well as possible factors contributing to the spread of HBV (ie tattoos, circumcision, etc.) were compiled from questionnaires. Twenty-one of the 25 (84%) HBsAg positive mothers had anti-HBe and none had HBeAg. One case of vertical hepatitis B virus transmission was seen while horizontal transmission during the study period occurred in two infants and in two older siblings. Potential risk factors for non-perinatal transmission were highly prevalent in both groups and may represent the main route of transmitting the infection, while, as shown in the present study, the vertical transmission of HBV infection only plays a minor role in the maintenance of the high infection rate in Ethiopia. Vaccinating all babies soon after birth would, therefore, be an effective means of eradicating or reducing the hepatitis B virus infection and its sequelae.

Serological and demographic survey of Epstein-Barr virus infection in Ethiopia.

A demographic and serological survey of Epstein-Barr virus infection was carried out in 5 geographically representative regions of Ethiopia. 80% of the 500 people studied were under 15 years of age. 82% of children under 5 years of age and 94% under 10 years of age were positive for IgG anti-viral capsid antigen (VCA) antibody. 51 of 100 children under 12 months of age and from 5 different provinces were positive for anti-VCA antibody. Of these, 23 were under 6 months. The distribution of anti-VCA antibody positivity was comparable in both sexes, in each age group and in the different provinces at different altitudes. Economic status, expressed in terms of estimated income, type of water supply, mode of excreta disposal and family size, did not significantly influence the distribution of anti-VCA antibody. Thus, early exposure to Epstein-Barr virus, with asymptomatic or subclinical presentation, probably accounts for the rarity of typical infectious mononucleosis in young adult Ethiopians. The lack of a relationship between Epstein-Barr virus infection and Burkitt's lymphoma, nasopharyngeal carcinoma, malaria as well as liver diseases is briefly discussed.

Insulin-dependent ketosis-resistant diabetes in Ethiopia.

Anthropometric, clinical and biochemical findings were compared in 30 rural (group A), 18 urban insulin-requiring (group B) and 45 urban oral-agent-responsive (group C) newly diagnosed diabetics. Mean ages at onset were 28.3 +/- 12.0, 25.6 +/- 14.5 and 42.1 +/- 10.5 years respectively. The differences between A and C and between B and C were significant. Group A were poor and malnourished, with body mass index (BMI) 15.9 +/- 1.9 and 17.2 +/- 3.7 kg/m2 for males and females respectively, presented with a long history of classical diabetes without ketoacidosis and required insulin in modest doses. 3 of 10 cases had excess stool fat but none of 13 unselected cases had pancreatic calcification. Group C were better nourished, with BMI 22.6 +/- 2.8 and 22.4 +/- 4.5 kg/m2, and responded to oral agents. Group B, with BMI 17.2 +/- 2.6 and 18.6 +/- 3.1 kg/m2, required insulin for control but had C-peptide levels above 0.02 nmol/1 in 10 of 15 cases. Anthropometric indices for males, but not for females, were significantly lower in group A than in group B or C. There were significant differences in levels of glucose between A and B and A and C, free fatty acids between A and C and B and C, insulin between A and B and A and C and C-peptide between A and C and B and C. Of the 3 groups the rural type most closely resembled the tropical variants.

Cryptosporidiosis among medical patients with the acquired immunodeficiency syndrome in Tikur Anbessa Teaching Hospital, Ethiopia.

Fresh stool specimens, collected at random from 63 medical in-patients with acquired immunodeficiency syndrome (AIDS), were studied prospectively for Cryptosporidium oocyst. The diagnosis of AIDS was made according to the clinical case definition of the Bangui criteria. These patients presented with profuse watery diarrhoea, significant weight loss and other associated symptoms and signs of clinical manifestations of symptomatic human immunodeficiency virus (HIV) infection. Using the modified Kinyoun acid fast staining technique, 25(39.7%) of the stool specimens were positive for Cryptosporidium oocyst. This study showed that the protozoan, Cryptosporidium parvum, may be responsible for a significant proportion of cases of chronic diarrhoea among AIDS patients in Ethiopia.

PIP: All adult patients who were admitted into hospital with chronic diarrhea and who fulfilled the modified World Health Organization surveillance case definition for AIDS (Bangui definition) were included in this study. The presence of generalized Kaposi's sarcoma or cryptococcal meningitis are sufficient by themselves for the diagnosis of AIDS surveillance purposes. Freshly passed liquid stool was collected from each patient. There were a total of 63 patients, 40 males and 23 females, 18-49 years old with a mean of 31 years. 42 (75%) of the 63 were residents of Addis Ababa city. 20 (31%) patients died in hospital. The duration of diarrhea varied from just a month to 9 months, with a mean of 4 months. The stool was watery in the majority of the patients except in those with amoebiasis and bacterial enteropathogen in whom blood and/or mucus was also present. Weight loss was dramatic and reached as much as 30-40% of body weight in some. 25 (39.7%) of study patients had oocyst of Cryptosporidium in their stools. Stool tests for other ova and parasites showed 42 (77%) to be negative. 21 (33%) were positive for single and mixed infections. Entamoeba histolytica accounted for 15 (11%), while Giardia lamblia and Strongyloides stercolaris were positive in 3 patients. Ascaris lumbricoides and hookworm were present concomitantly in 6 of the cases with protozoal parasites. 21 patients had stool cultures that grew no enteropathogens. In 6 others, cultures grew Salmonella, Shigella and Campylobacter species in 4, 2, and 2 respectively. Double infections were observed in 2 cases. Enteropathogenic parasites and microbial agents were identified in about 63% of patients. In the absence of effective antibiotic or chemotherapeutic agents for the treatment of protozoan enteropathogen, the stool smear for Cryptosporidium adds extra cost to the management of AIDS. Prevention remains the only alternative, both for AIDS and cryptosporidiosis.

The efficacy of norfloxacin in the treatment of urinary tract infections in Tikure Anbessa Hospital, Addis Ababa, Ethiopia.

The efficacy of norfloxacin 400 mg every 12 hours for 7 days was prospectively and openly studied among patients with urinary tract infection. Enrolled were 30 patients based on inclusion and exclusion criteria. 23 patients completed the study and were evaluated for efficacy and tolerability with urinary infections defined as a bacterial count of at least 10(5) bacterial per ml of urine. Follow-up urine cultures at 14-21 days and 4-6 weeks post-treatment were done. 21 of 23 were cured and this was a cure rate of 91%. There were no clinical adverse effects which necessitated cessation of treatment. In vitro test studies were carried out to compare the activity of norfloxacin with commonly used antimicrobials as well as to observe its activity against other organisms which are not normally urinary pathogens. In this aspect of the study, norfloxacin was found to be superior to eleven other antimicrobials with which it was compared. This study showed that drug is very active against urinary and enteric pathogens.

Heritability of diabetes mellitus in Ethiopian diabetics.

A prospective case control study was conducted in 859 diabetic probands and 1059 nondiabetic controls. These were interviewed for history of diabetes mellitus among their first degree relatives. The protocol also included second and third degree relatives. The prevalence of diabetes mellitus in the first degree relatives of cases and controls showed a statistically significant difference (Z = 6.7564 and P = 0.00000). Similarly, the prevalence of diabetes among the second and third degree relatives of cases and controls differed significantly (Z = 2.74, P = 0.006). There were 445 non-insulin dependent diabetes mellitus (NIDDM) and 414 insulin dependent diabetes mellitus (IDDM) in the diabetic probands. There was more family history of diabetes mellitus among the first degree relatives of NIDDM than IDDM and the difference is statistically significant (Z = 4.076, P = 0000). The second and third degree relatives of NIDDM and IDDM also differed significantly (Z = 4.05, P = 00005). This study indicates that heredity plays an important role in the genesis of diabetes mellitus in Ethiopian diabetics.

Crohn's disease: report of seven cases from Ethiopia.

Crohn's disease is rare in Africa. We present the clinical features, surgical management and the histopathological findings of surgical specimens of seven cases of Crohn's disease.

Idiopathic ulcerative colitis among Ethiopian patients with chronic diarrhoea.

Using both exclusion and inclusion criteria, a search for idiopathic ulcerative colitis was made among patients who presented with chronic diarrhoea over a period of 3 years. Five of 19 patients with chronic diarrhoea were clinically, histologically and radiologically compatible with idiopathic ulcerative colitis. Though rare, idiopathic ulcerative colitis does exist among this population and, therefore, should be looked for in patients presenting with chronic diarrhoea.

Familial polyposis in two Ethiopians.

Cases of familial polyposis coli and Peutz-Jeghers syndrome are reported for the first time in Ethiopia. One case seemed to have the defect as a new mutation in his gene while the other possibly inherited from his father. The one with polyposis coli had transmitted the disease to his offspring. This patient had total colectomy for prophylaxis against a potential carcinoma of the colon.

The role of milk and lactose intolerance in Ethiopian patients with non-ulcer dyspepsia: a case control study.

Milk intolerance, lactose intolerance and non-ulcer dyspepsia are common among Ethiopians. This study, therefore, was designed to find out if milk intolerance associated with lactase deficiency account for non-ulcer dyspepsia. Ninety-eight patients with non-ulcer dyspepsia and 95 controls were examined and interviewed for demographic data and milk drinking habits. Then each had a lactose tolerance test (LTT), stool examination for pH, ova and parasites. The demographic characteristics and the number of milk drinkers were comparable in the 2 groups. However, milk intolerance and lactose intolerance were significantly higher among the patients with non-ulcer dyspepsia than among the control group (p less than 0.01, p less than 0.05 respectively). The combination of milk intolerance, lactose intolerance and LTT was also significantly different (p less than 0.01). The mean stool pH was markedly reduced after lactose ingestion and there were more ova and parasites in the stools of the control group. These observations suggest that milk intolerance and/or lactose intolerance account significantly for the symptoms of the patients with non-ulcer dyspepsia. However, since lactose intolerance and abnormal LTT are very common among adult Ethiopians symptoms related to the drinking of milk should be interpreted with caution vis-a-vis the results of the lactose loading test.

Serological survey of human immunodeficiency virus infection in Ethiopia.

PIP: A serological survey was conducted on 1754 people in various parts of Ethiopia from 1982-1987, with no HIV positive findings until 1984. The problem of Aids reached epidemic proportion in North America and Central Africa in the early 1980s. The reasons for delayed appearance and the source of HIV infection in Ethiopia are not clear. There were 4 groups of subjects tested. The 1st from 1982-83 consisted of 500 patients: 100 each from Asmara, Harar, Addis Ababa, Yirga Alem, and Nekemte; the 2nd in 1983 consisted of 459 volunteer blood donors in Addis Ababa; the 3rd in 1984 consisted of 167 patients with Bell's palsy, and 100 others; the 4th from 1985-87 consisted of 528 patients of the liver clinic of Tikur Anbessa Hospital. The 1st 2 HIV positive sera appeared in group 3 during 1984, and since then 13 patients examined for liver and gastrointestinal conditions were found to be positive. It may be speculated that the new and delayed introduction of HIV infection into Ethiopia is part of the transcontinental spread, caused by migration of refugees, frequent visits to existing harbors by sailors, and the influx of many international organizations to Addis Ababa where prostitution is common. Considering the high prevalence of HIV infection among prostitutes, promiscuity might be the major mode of transmission in this population. The multiple use of inadequately sterilized hypodermics and blood transfusions without screening also contribute to the spread of HIV infection.^ieng

Chronic liver disease in Ethiopia: a clinical study with emphasis on identifying common causes.

Between July 1986 and April 1989, 334 hospitalized adult Ethiopian patients with chronic liver disease were studied according to a protocol to define their clinical features and to identify risk factors with the aim of preventive intervention. Of these, 14 had chronic hepatitis, 208 cirrhosis and 112 hepatocellular carcinoma (HCC). Both clinical and histological diagnostic criteria were employed. A detailed questionnaire was used to document demographic and clinical data. A common clinical presentation among patients with chronic hepatitis was darkening of the face and hands with or without hypertrichosis of the face and blisters over the dorsi of the hands. This overt or latent form of porphyrea cutanea tarda (PCT) responds to chloroquine. Patients with cirrhosis of the liver commonly present for the first time with ascites, splenomegaly, haematemesis and/or melena from oesophageal varices, and mental changes due to hepatic encephalopathy. Overt or latent forms of PCT are also common features. Peculiar to these cirrhotics is the rarity of spider naevi, gynaecomastia, testicular atrophy, Dupuytren's contracture, parotid gland enlargement and clubbing of the fingers. Exhaustion, loss of appetite, rapid loss of weight, right upper quadrant and/or epigastric pain (all often of less than 6 months' duration, a big, hard, tender and grossly nodular liver with bruit, signs of portal hypertension, and/or hepatic encephalopathy, in a young male with a rapid down hill course characterize the Ethiopian patient with HCC. Serum anti-nuclear factor, anti-mitochondrial anti-bodies and anti-smooth muscle anti-bodies were absent in those with chronic hepatitis and were uncommon in the cirrhotics and HCC cases. One or more hepatitis B virus markers were found in 86% of chronic hepatitis, 88% cirrhosis and 78% HCC and the HBsAg carrier state was found in 36%, 29% and 23%, respectively. Among the HBsAg carriers, HBeAg positivity was less common than anti-HBe but anti-HDV was significantly higher than in the healthy general population. Alphafetoprotein (AFP) levels greater than 500 mg/ml were present in 16 (8%) cirrhotics and 58 (52%) patients with HCC. Histologically, 3 of the chronic hepatitis patients had progressed to cirrhosis, 8 of the cirrhotic patients had chronic active hepatitis and 85% of HCC cases occurred in a background of macronodular cirrhosis. Three cirrhotics developed HCC during follow-up.(ABSTRACT TRUNCATED AT 400 WORDS)