SEROPREVALENCE OF HAV, HBV, HCV, AND HEV AMONG ACUTE HEPATITIS PATIENTS AT KENYATTA NATIONAL HOSPITAL IN NAIROBI, KENYA.

BACKGROUND: Acute viral hepatitis is most frequently caused by the hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis E virus (HEV). OBJECTIVES: To determine seroprevalence of HAV, HBV, HCV and HEV among patients with acute hepatitis in Nairobi, Kenya, elucidate various risk factors for hepatitis viral infection and determine the co-infection rates with these viruses in the acute hepatitis patients. DESIGN: Across sectional descriptive study. SETTING: Kenyatta National Hospital, from November 2007 to April 2008. SUBJECTS: One hundred patients were recruited by purposive sampling method and comprised of 57 males and 43 females. RESULTS: Among the enrolled patients, twenty three tested positive for one or more markers of acute viral hepatitis, that is, HAV, HBV, HCV and HEV. No markers were detected in 77patients, 2% tested positive for IgM anti-HAV 11% for IgM anti-HBc; 3% for HBsAg; 5% for HCV RNA and 7% for IgM anti-HEV.Various risk factors associated with acute viral hepatitis were identified; poor sanitation, source of water, occupation, place of residence, level of education, household size, drug abuse and sexual behaviours. Co-infection rate with hepatitis Viruses was at 4%, IgM anti-HAV and IgM anti-HEV 1% (n=1); IgM anti-HBc and IgM anti-HEV 1% (n=1); IgM anti-HBc and anti-HCV 2% (n=2). Three patients were positive for HBsAg; among this two were negative for IgM anti-HBc and this accounted for HBV carriage (2%). CONCLUSION: Hepatitis viruses'infections are commoncause of hepatitis among patients with acute hepatitis at Kenyatta National Hospital. Co-infection with these viruses was also identified among these patients.

The detection of non-RoTat 1.2 Trypanosoma evansi.

The majority of Trypanosoma evansi can be detected using diagnostic tests based on the variant surface glycoprotein (VSG) of Trypanosoma evansi Rode Trypanozoon antigen type (RoTat) 1.2. Exceptions are a number of T. evansi isolated in Kenya. To characterize T. evansi that are undetected by RoTat 1.2, we cloned and sequenced the VSG cDNA from T. evansi JN 2118Hu, an isolate devoid of the RoTat 1.2 VSG gene. A 273 bp DNA segment of the VSG gene was targeted in PCR amplification for the detection of non-RoTat 1.2 T. evansi. Genomic DNA samples from different trypanosomes were tested including 32 T. evansi, 10 Trypanosoma brucei, three Trypanosoma congolense, and one Trypanosoma vivax. Comparison was by PCR amplification of a 488 bp fragment of RoTat1.2 VSG gene. Results showed that the expected 273 bp amplification product was present in all five non-RoTat 1.2 T. evansi tested and was absent in all 27 RoTat 1.2-positive T. evansi tested. It was also absent in all other trypanosomes tested. The PCR test developed in this study is specific for non-RoTat 1.2 T. evansi.

PCR amplification of RoTat 1.2 VSG gene in Trypanosoma evansi isolates in Kenya.

A direct card agglutination test for Trypanosoma evansi, CATT/T. evansi based on the predominant variable antigen-type (pVAT) RoTat 1.2 was evaluated previously in the field in Isiolo District, Kenya. Sixteen out of 51 (31.4%) parasitologically positive camels were negative by the antibody detection test. In the present study, trypanosomes isolated from the camels were analysed in an attempt to determine the cause of the false negative results of CATT/T. evansi. A total of 20 field isolates comprised 16 stocks from camels that were negative by CATT/T. evansi, and 4 from CATT/T. evansi-positive camels. In addition, 15 known T. evansi and four T. brucei were used as reference. Purified DNA samples were tested using an established RoTat 1.2-based polymerase chain reaction (PCR) that yields a 488 bp product for the specific detection of T. evansi. Antibodies to RoTat 1.2 variant surface glycoprotein (VSG) were used in Western blotting to detect RoTat 1.2 VSG linear epitopes. Results of PCR and Western blot showed that the 16 stocks isolated from CATT/T. evansi-negative camels fell into three groups. In Group 1, both the RoTat 1.2 VSG gene and the VSG were absent in three stocks. In five trypanosome stocks in Group 2, the RoTat 1.2 VSG gene was detected, but Western blot was negative indicating absence of the expressed VSG. Five other stocks containing the RoTat 1.2 VSG gene were also in this group. The RoTat 1.2 VSG gene was detected and Western blot was positive in all four trypanosome stocks in Group 3. All four stocks from CATT/T. evansi-positive camels contained the RoTat 1.2 VSG gene and the expressed VSG. The reference T. evansi KETRI 2479 lacked the RoTat 1.2 VSG gene and there was no immune reactivity detected by Western blot. The rest of the reference T. evansi stocks examined contained the RoTat 1.2 VSG gene. All the four T. brucei samples examined were negative by PCR and Western blot. In conclusion, this study showed that the RoTat 1.2 VSG gene was absent from some T. evansi trypanosomes in Kenya.

Evaluation of antigen and antibody rapid detection tests for Trypanosoma evansi infection in camels in Kenya.

The card agglutination test for Trypanosoma evansi (CATT/T. evansi) for the detection of antibodies, and Suratex for the detection of circulating antigens were compared in a cross-sectional study involving camels in eastern and central parts of Kenya. Of the 2227 camels screened, 2038 were owned by nomadic pastoralists in T. evansi endemic areas in eastern Kenya. A herd of 86 camels were from a ranch in Mugwoni. In Athi River area, 35 camels belonged to Kenya Trypanosomiasis Research Institute, and 68 were slaughter animals. Diagnostic sensitivity estimates were obtained by testing sera from 51 camels that had been found to be parasitologically positive by the haematocrit centrifugation technique, buffy-coat technique and mouse inoculation. Diagnostic specificity was estimated by testing sera from 35 camels known to be trypanosome-free. Positive and negative predictive values (NPVs) were calculated using a range of prevalence values. The sensitivity of CATT/T. evansi (68.6%) was higher than that of Suratex (58.8%), but not significantly. Both tests had equally high specificity (100%). The overall prevalence was 2.3% (51 out of 2227) by parasite detection, 32.2% (327 out of 1017) by CATT/T. evansi and 19.6% (188 out of 961) by Suratex. Overall, there was a positive association between CATT/T. evansi and Suratex though the strength of association was low (McNemar's test=46.12, P=0.001; kappa=0.26, CI: 0.20-0.33). Parasite prevalence ranged from 0% in several herds to 27.8% in a herd in Isiolo. Prevalence was highest in Isiolo with 2.5% (51 out of 2030) by parasitological detection, 38.8% (321 out of 828) by CATT/T. evansi and 21.9% (169 out of 772) by Suratex. In Mugwoni prevalence was 7 and 18% by CATT/T. evansi and Suratex, respectively, and no parasites were detected. In Athi River Suratex detected 2.9% (3 out of 103) positive while CATT/T. evansi and parasitological methods gave negative results. At prevalence values between 10 and 100%, CATT/T. evansi as well as Suratex had infinitely high positive predictive values, whereas Suratex had a lower NPV than CATT/T. evansi. In conclusion, results of this study showed that CATT/T. evansi and Suratex were able to detect aparasitaemic infections rapidly and were more sensitive than parasitological methods in revealing the true extent of trypanosomosis in a herd. The tests effectively complemented parasitological methods in the detection of T. evansi infections in camels.

Animal-level risk factors for Trypanosoma evansi infection in camels in eastern and central parts of Kenya.

Point prevalences and animal-level risk factors for Trypanosoma evansi infection were investigated in a cross-sectional study that involved 2227 camels from eastern and central parts of Kenya. The screening tests used were haematocrit centrifugation technique (HCT), mouse inoculation and latex agglutination (Suratex). All camels were screened with HCT, while 396 and 961 of them were, in addition, screened with mouse inoculation and Suratex tests, respectively. Parasitological and Suratex test results were used in parallel to determine the number of camels exposed to T. evansi infections. Statistical analyses were conducted using Statistical Analysis Systems. Parasitological and Suratex test results in parallel were dependent variables in multivariable logistic regression models that determined risk factors for T. evansi infection. Herd-level clustering was corrected with general estimation equations. The prevalences were 2.3% and 19.6%, using parasitological and Suratex tests, respectively, and 21.7% when both tests were used in parallel. There was a positive association between the screening tests (McNemar's test = 104.8, P = 0.001) although the strength of association was low (Kappa = 0.2; 95% CI: 0.1-0.3). Before accounting for herd-level clustering, dry season (OR = 1.5; 95% CI: 1.0, 2.1) and nomadic pastoralism (OR = 1.8; 95% CI: 1.1, 3.2) were associated with increased odds of a camel being exposed to T. evansi infection compared to wet season and ranching, respectively. Following this correction, only nomadic pastoralism was significantly associated (OR = 3.1; 95% CI = 1.0, 14.4) with T. evansi infection compared to ranching. It is concluded that camels managed under nomadic pastoralism had higher risk of being exposed to T. evansi infections than camels from ranching systems of management.

Immunoassay of circulating trypanosomal antigens in sleeping sickness patients undergoing treatment.

Sera from 99 patients infected with Trypanosoma brucei rhodesiense and undergoing treatment, were analyzed for circulating trypanosomal antigens using a sandwich antigen-trapping enzyme-linked immunosorbent assay (ELISA). Trypanosomal antigens were detected in 83 (84%) of the patients. Post-treatment antigen profile in 67 patients showed five distinct patterns: in 48% of the patients antigen levels remained elevated throughout the time of hospitalisation and follow-up; in 31%, antigens dropped to the negative value by the second month; in 7.5%, antigens dropped to the negative level and became elevated afterwards; in 7.5%, antigen levels were negative initially, but later, became elevated and remained so throughout the observation period; in 6%, antigen levels remained below the negative value throughout. All patients who relapsed on follow-up had earlier shown evidence of elevated antigen profile. There were no cases of relapses among 21 patients whose antigen levels dropped subsequent to treatment. This ELISA trypanosome antigen detection test could be useful in evaluating treatment success, when used together with parasitological diagnostic techniques.

Evaluation of Procyclic Agglutination Trypanosomiasis Test (PATT) for the immunodiagnosis of Trypanosoma brucei rhodesiense sleeping sickness in Kenya.

Documented sera from 156 patients admitted to Alupe Sleeping Sickness Hospital in Western Kenya were tested to determine the potential usefulness of Procclic Agglutination Trypanosomiasis Test (PATT) for the diagnosis of Trypanosoma brucei rhodesiense African sleeping sickness. A total of 490 serum samples were tested, including 42 controls. Anti-trypanosome antibodies were detected in 99% of the sera taken prior to trypanocidal drug therapy. Antibody levels remained high during course of treatment. In cured cases antibodies declined to negative or low levels 4 months to one year after treatment. High antibody levels persisted in patients who relapsed. Although the results showed a high sensitivity and specificity, confirming the potential usefulness of the test for serodiagnosis of African sleeping sickness, PATT, in its present form is unsuitable for routine diagnosis. This is due to difficulties inherent in the use of live trypanosomes as detector antigen.

Circurating trypanosomal antigen levels in sleeping sickness patients under going treatment at Alupe

Sera from 99 sleeping sickness patients admitted to Alupe Hospital were analyzed for circulating trypanosomal antigens using a sandwich antigen-trapping enzyme immunosorbent assay. Trypanosomal antigens were detected in 83 (84) of the patients. Post-treatment antigen profile in 67 patients showed five distinct patterns: in 48 of the patients antigen levels remained elevated thoughout; in 31 of the patients antigens dropped to the; negative value; and became elevated afterward; in 6.0 of the patients antigen levels were negative initially; but became elevated later; in 7.5 of the patients antigen levels remained below the negative value thoughout. The significance of these observations in the clinical management of sleeping sickness is discussed

Phagocytosis of antibody-sensitized Trypanosoma brucei in vitro by bovine peripheral blood monocytes.

The interaction between bovine peripheral blood monocytes and Trypanosoma brucei organisms in the presence of immune whole serum or specific purified IgM or IgG1 antibodies from infected cattle was investigated. Adherence of antibody-sensitized trypanosomes to freshly isolated monocytes from non-infected animals was not observed, confirming the absence of receptors for IgM and IgG1 on fresh monocytes. After in-vitro cultivation of the monocytes for at least 3 hr, adherence of IgM-sensitized trypanosomes was consistently demonstrable, while the adherence of IgG1-sensitized trypanosomes was not observed until the monocytes had been cultured for 7 days. The adherence of IgM-sensitized trypanosomes occurred in the absence of bovine complement and was Ca++-dependent, indicating that the interaction between sensitized trypanosomes and bovine monocytes was mediated through a specific combining site for monocytes in the Fc region of the IgM molecule rather than through a C3b receptor. In contrast to the monocytes from uninfected animals, freshly isolated peripheral blood monocytes from T. brucei- or T. congolense-infected cattle were capable of binding IgM- or IgG1-sensitized trypanosomes after only 30 min and 24 hr of in-vitro cultivation, respectively, suggesting that peripheral blood monocytes from infected animals were already in an "activated' state. Electronmicroscopic examination revealed that IgM- or IgG1-sensitized trypanosomes were rapidly engulfed and digested by the monocytes.

Analysis of the variable antigen composition of Trypanosoma brucei brucei metacyclic trypanosomes using monoclonal antibodies.

The metacyclic trypanosomes of a Trypanosoma brucei brucei clone (ILTat 2.1) were analysed with regard to their variable antigen (VAT) composition using monoclonal antibodies. The metacyclic population was antigenically heterogeneous. Despite the heterogeneity, however, the overall VAT composition of the metacyclic population appeared to be limited in number. A similar pattern of reactivity was observed when the monoclonal antibodies were tested on metacyclics of another clone (IL Tat 2.2) derived from a rabbit 30 days after infection with IL Tat 2.1 as well as those of the parent stock (STIB 247). The VAT characteristics of the metacyclics of this serodeme were consistent regardless of whether they were transmitted by Glossina morsitans morsitans or G.m. centralis. The monoclonal antibodies also reacted with some of the bloodstream VATs isolated within 72 h from cyclically infected mice. None of the monoclonal antibodies, however, reacted with metacyclics of a different stock (LUMP 227).

Immune depression in African trypanosomiasis: the role of antigenic competition.

The capacity of trypanosome-infected cattle to mount an immune response to a simultaneous or subsequent challenge with other trypanosomes was investigated using various clones of Trypanosoma congolense and T. brucei. In animals infected simultaneously with equal numbers of trypanosomes of two different clones, the variant-specific antibody response to one clone was severely depressed while the response to the other was not affected. In cattle infected with one clone and then subsequently challenged severely depressed depending on the time interval between the two inoculations. These observations were consistent regardless of whether the clones of trypanosomes used were derived from the same or different species. The characteristics of these responses would suggest that the inability of trypanosome-infected cattle to respond well to a simultaneous or subsequent challenge with other trypanosomes or other antigens may be due to antigenic competition.