Evaluation of Pcr Assay for Hepatitis C Virus in Peripheral Blood Mononuclear Cells Of

Journal of Babylon University/Pure and Applied Sciences/ No.(9)/ Vol.(22): 2014

Frequency of V. cholerae in Babylon Province

Raed Fanoukh Alaouadi

College of Veterinary Medicine /Al-Qasim Green University ,Iraq

Mobile:009647809430916

Abstract:

Background: Cholera is a severe and sometimes lethal human diarrheal disease caused by

the non- invasive gram-negative bacterium Vibrio cholerae. In the last decades, there is change in the disease epidemiology specially in the risky group and till now there is difficulty in preparation of effective vaccination programs.

Aim of study: to study the epidemiology together with the virulent factors in hope of future effective vaccine preparation and also to assess the ability of introduction of helpful laboratory monitoring parameter to assess the clinical disease.

Materials and Methods: in this study, a total of 21550 patients who complained of diarrheal disease of whatever cause during 1/8-31/12/2008 were enrolled and majority consulted Primary Health Care Centers or admitted to hospitals. Stool samples were taken and transported with Cary & Blair transport medium for culturing on specific media and further identifications. All isolates were proved to be vibrio cholerae in Central Public Health Laboratory, Baghdad. Epidemiological parameters were studied in addition to virulent factors(haemagglutinin, TCP(to study autoagglutination) and protease (to assess disease dissemination).

Results: this study showed that the incidence of Vibrio Cholerae in Babylon province was (1.07 % ). The commonly affected age group by Vibrio Cholerae was ( 1-5 years) (53.08%).The only detectable serotype of this pathogen was Vibrio Cholerae O1, biotype El Tor, serotype Inaba. Autoagglutination were(mild: 12.75%; moderate; 21.81% and high: 65.43%) while haemagglutination was (74.19%, 92.45% and 96.85%) in isolates which showed mild, moderate and high autoagglutination respectively. Protease was raised in those with late severe cholera in comparison to early mild cholera.

Conclusions: Re-emergence of cholera is highly anticipated. V. cholera have a major virulent colonization factors in those with severe cases and less in mild cases. Live attenuating vaccine against major colonization factor through artificial mutation to gene of interest might be preperared through use of virulent factor gene as target in future. Protease might be helpful as a laboratory monitoring parameter to assess cholera severity and dissemination.

Keywords: V.cholerae, cholera vaccination, epidemiology of cholera

الخلاصة

الخلفية: مرض الكوليرا هو مرض شديد ويعتبر مميت في بعض الاحيان تسببه ضمات الكوليرا وهي جراثيم سالبة الغرام وحيث انه في الآونة الاخيرة ثبت وجود تغيير في وبائية المرض وخصوصا في مجاميع الخطورة حيث انه توجد صعوبات في تحضير لقاح مضاد فعال.

الهدف: تهدف الدراسة لتقييم عوامل ضراوة ضمات الكوليرا مع دراسة وبائية المرض املا لتحضير لقاح فعال في المستقبل وكذلك لتقييم امكانية استخدام احد عوامل الضراوة كمعيار لتقييم المرض السريري مختبريا.

المواد وطرق العمل: تم استحصال 21550 نموذج براز لمختلف حالات الاسهال والتي كان معظمها من المراكز الصحية والمستشفيات للفترة من 1/8-31/12/2008ونقلت هذه النماذج بواسطة وسط ناقل لغرض زراعتها وتشخيصها مختبريا واحيلت النماذج الموجبة الى المختبر المركزي في وزارة الصحة حيث تم توكيد تشخيصها. تمت دراسة بعض العوامل الوبائية وعوامل الضراوة باستخدام طرق مختبرية لدراسة التلازن الذاتي و التلازن الدموي وانزيم البروتيز لقياس امكانية استخدامه كمعيار مختبري لانتشار الجرثومة في الجسم.

النتائج: تبين ان نسبة حدوث مرض الكوليرا في محافظة بابل هو(1.07%) وان اكثر الفئات العمرية اصابه هي الاعمار (1-5 سنوات) وان النوع المصلي الذي تم تأكيده هو من نوع الانابا وتبين كذلك بأن نسب التلازن الذاتي كانت(12.75% للحالات البسيطة و 21.81% للحالات المتوسطة و65.43 % للحالات العالية) وتبين بأن نسبة أنزيم التلازن الدموي هو (74.19% و92.45% و 96.85%) للعزلات التي تبدي تلازن ذاتي بسيط ومتوسط وعالي على التوالي ووجد كذلك بأن أنزيم البروتيز يكون بمستويات عالية في الحالات الشديدة مقارنة بالحالات البسيطة.

الاستنتاج: يستنتج بأن توقعات عودة مرض الكوليرا عالية جدا وكذلك تبين بان ضمات الكوليرا تمتلك عوامل ضراوة رئيسية خاصة بالاستيطان وخصوصا في الحالات الشديدة ومن خلال عمل طفرات وراثية مصطنعة للجين المطلوب والمسؤول عن الاستيطان يصبح بالإمكان تحضير لقاح مستقبلا. كما تبين انه بالإمكان استخدام انزيم البروتيز كمعيار مختبري لمتابعة شدة وانتشار حالات الكوليرا.

كلمات مفتاحية : ضمات الكوليرا ، لقاح الكوليرا ، وبائية الكوليرا

Introduction:

Cholera is an acute enteric infection caused by the ingestion of bacterium Vibrio cholerae present in faecally contaminated water or food (WHO,2007) Cholera spreads mainly via drinking water supplies contaminated by human excreta, especially from sub clinical carriers and mild cases(Al–abbassi A.M,2005). Cholera is endemic in Iraq, and WHO estimates that up to 600 cases of cholera occur in Iraq annually. Following the cholera outbreak in 2007, the Ministry of Health, with the support of WHO has established 950 surveillance sites in Iraq. These sites report every two weeks an acute watery diarrhea and cholera cases(HNSOT report,2008). The study of any microorganism’s properties can be greatly enhanced by the generation of mutation in genes of interest and subsequent genetic mapping can elucidate the identity, relative size, number and organization of genes involved in a physiological process that can define the recognition and location of transcriptional units that is used to create strains with desired proprieties, to overproduce a desired metabolite or enzymes and vaccines development (Calam, C. T. 1970 & Dougan, G et al, 2002).In the last decades, attention to cholera epidemiology increased, as cholera epidemics became a worldwide health problem. Detailed investigation of V. cholerae interactions with its host and with other organisms in the environment suggest that cholera dynamics are much more complex than previously times (Codeco, C.T. 2001) ,so this study aimed to study the epidemiology together with virulent factors in hope of future vaccine preparation to achieve effective control and also to assess the ability of introduction of helpful laboratory monitoring parameter to assess the clinical disease.

Materials and Methods:

Specimens: Sampling was carried out from August to December 2008. About 22557 stool samples (mucus-flecks from stools) or rectal swabs (Jawetz, M. 1998 & Sack D. 2004) were collected from all patients admitted to all Babylon Hospitals with all patterns of diarrhea in additional to stool samples from their relatives and those that consults primary health care centers in the Babylon during the period (1/8-31/12/2008) to diagnose Vibrio's cases and to detect carrier state according to Iraqi Ministry of Health instructions. Samples were collected before antibiotic therapy administration. Personal data such as name, age, sex, and location, date of onset of disease, suspected disease and type of treatment received, if any, were collected.

Transport, enrichment and plating:

All the samples were collected into Cary-Blair transport medium (13.5 g /liter; pH 8.4; Oxoid; England) (Tison, D et al 1999) and delivered to the Regional Hospitals where further investigations in form of standard microbiological methods used for the culture and identification of V. cholerae isolates .Samples were enriched in alkaline peptone water broth (APW) (10 g/liter; sodium chloride, 10 g/liter; pH 8.6 Himedia, India) at 37°C for 6 to 8 h before plating (Huq, A., R 1995 et al & Islam, M. S 1994).

Samples processing and Culture procedure (R3)

A loopful of the culture broth(5 μl), was taken from the top layer of the APW then streaked onto Thiosulfate-Citrate-Bile salts-Sucrose (TCBS) (89 g/liter ; PH 8.6 ± 0.2, Rashmi Banglor ; India), MacConkey agar (51.5/liter, Himedia ; India) and blood agar ( 40.0 g/liter , Rashmi Banglor ; India) and incubated at 37°C for 18 to 24 h. Two-three colonies with the characteristic appearance (2 to 3 mm in diameter, yellow, and flat) were subjected for further identification. Any patient with negative stool culture would be excluded from the study(Oliver, J. D. and Kaper, J. B. 1997& Collee, J.G et al 1996).

The isolates were further identified by

1. Biochemical identification done according to ( Collee, J.G et al 1996& Perilla, M.J.,2003).:

a. Oxidase test: This test was done according to (Baron, E.J,1994). Oxidase-positive strains were further identified with commercially available serological tests and with the API 20 E system.

b. String test :was used to differentiate Vibrio species (spp. ) from Aeromonas spp and plesiomonas shigelloides and done especially for cultures that resemble V cholerae but fail to agglutinate in diagnostic antisera. . The string test done according to(Murray P.R et al ,1995& Forbes B A et al,2007).

c. API-20 E: Also the isolates confirmed biochemically by using API 20-E system (Biomereumix / France) according to instructions of supplied company.

2-Biotyping

The biotype of V. cholerae serogroup O1 strains was distinguished by the following methods; polymyxin B susceptibility, hemolysin test, and Voges-Proskauer test(Elliot, E. L et al ,2001& Butterton, JR and Calderwood, SB.,1995).

3-Serotyping

Serologic confirmation was done using polyvalent, anti-Ogawa, anti-Inaba antisera ( Plasmatec Laboratory Products Ltd) (Plasmatec/ UK)( Elliot, et al, 2001) and O139 antisera (GlaxoWellcome, England) (Tison et al,1999 & Kay et al 1994)). serotyping according to Baumann and Schubert (Baumman and Schubert, 1984); Elliot et al.( Elliot,2001); and WHO (WHO.2002).Cultures that resemble V. cholerae but fail to agglutinate in diagnostic antisera (non agglutinable or non-O group 1 vibrios) will be subjected to additional tests such as decarboxylases and the "string test.

4- Virulent factor detection:

a. Haemagglutination Test (HA): RBC suspension was prepared from the human blood (group A). Human blood washed with phosphate buffer saline (repeated 3 times), 3% suspension from RBC (V/V) was then prepared and at the same time, blood agar was prepared, then it was inoculated with bacteria and incubated at 37C˚ for 24 hours, after that, place 1 drop of human red blood cells with loopful onto a clean slide, to this, drop added 1 drop of bacterial culture by using a flamed loop were mixed with human red blood cells on clean slide. The blood agglutination with bacteria was detected in room temperature during (1-5) minutes. Agglutinated red blood cells in suspension (positive reaction) had a clumped appearance distinct from non-agglutinated red blood cells (negative reaction) (Francis et al ,2002).

b. Detection of Toxin coregulated pili (TCP): Vibrio cholerae hydrophobicity increased in broth culture due to the expression of pili causing visible clumping of bacteria as a pellet at the bottom of tube and leaving a clear supernatant; this phenomenon is known as autoagglutination which detected by macroscopical examination (Schuhmacher and Klose1999).

c. Autoagglutination Phenomenon: AKI broth pH6.9 was inoculated with the mutant isolates and incubated at 37ºC for 18 hour in 110 rpm shaker incubator, autoagglutination was screened and compared with wild isolate of V. cholerae S cultured at the same conditions(Al- Khafaji, 2007 and Schuhmacher and Klose,1999 ).

d. production of Protease : V. cholerae were streaked on TSA containing either 1% casein or 1% tween 80 or 7% sheep blood and incubated for 18-48 hour at 37°C. Cleared zone was used to detect proteases(Al- Khafaji, 2007).

5. Confirmatory tests: after regional hospital diagnosis referral would be done to Central Public Health Laboratory, Babylon . All human isolates proven to be Vibrio cholerae in Central Public Health Laboratory/ Babylon were reviewed and approved by Central Public Health Laboratory, Baghdad/ Ministry of Health/Iraq.

Statistical analysis: Data were presented as percentages and the results were analyzed using SPSS version 14 statistical system. Student t test was used to compare between results. P value less than 0.05 was considered as statistically significant value.

Results:

Table (2): Incidence of Vibrio cholerae in Babylon Province according to the results of stool culture on (TCBS) agar

This table showed that V. cholera was isolated from all age groups and the most commonly affected age groups were children and the highest incidence was seen in children 1-5 years old age.

Table (2): Incidence of Vibrio cholerae in Babylon Province according to the results of stool culture on (TCBS) agar

This table showed that out of 22557 cultured stool samples only 243 samples give positive results with an incidence of about (1.07%) of all diarrheal cases.

Table(3): Frequency of Vibrio’s species in Babylon Province according to its biotypes and serotypes

This table showed that all 78.65 % of the encountered cholera cases are agglutinable vibrio and all agglutinable V. cholerae are of Inaba serotype while the non agglutinable V. cholerae account for 21.35%.

Table (4): Degree of autoagglutination of V. cholerae isolates

This table showed that most V. cholerae pathogens (about 65.43%) showed high autoagglutination.

Table (5): percentages of V. cholerae haemagglutination in regard to degree of autoagglutination

This table showed that most V. cholerae which showed moderate and high degree of autoagglutination have higher percentages of haemagglutination.

Table ( 6 ): protease activity in relation to phase of clinical presentation

Out of 243, 73 with early mild and 97 with late severe cholera (total =170) were chosen to study protease activity and the remaining ( 73 isolates) were not included in this test because they are inconsistent with the required classification in relation to time of onset and severity of the condition at the same time. A high percentage of protease activity presented in cases with late severe cases of cholera.

Discussion:

Table -1 showed that the most commonly affected age groups were children (< one year, 15.22%; 1-5 years ,53.08%). V. cholera was isolated from all age groups and the highest incidence was seen in children (1-5) years old age The results had highlighted the cholera occurrence in the age group less than five years which is inconsistent with the epidemiological definition of Vibrio cholerae. This might reflect the cause of the disease which might be due to poor sanitation and educational measures of this age groups especially the rural areas (Iredell and Manning,2004) in addition to decrease in vibriocidal antibody titers which increases with age and correlate with protection from cholera (Glass et al,1985). Since cholera is essentially a local disease of the small intestine, it would seem that local defense might be a main determinant of protection against infection by V. cholerae. Recurrent infections of cholera are ,in fact, rare and this is probably due to local immune defense mediated by antibodies secreted onto the surfaces of the intestinal mucosa. Moreover, in children who are nursing, cholera is less likely to occur presumably due to protection afforded by secretory antibody in mother's milk (Elliot. 2001).This is consistent with (Noaman,2011) and (AL-Naddawi and Khalid,2009) ,while it is inconsistent with (Al-Shok and Baey (2009) who noticed that the common age group was ranged from 12 – 65 years with a mean of 28.8 ± 12.5 years during the Epidemic of Cholera that was anticipated in Babylon Governorate, during the years 1998 and 1999. This variation between our study and (Al-Shok and Baey (2009) may be related to V. cholerae serotype as it V. cholerae serotype Inaba in our study and in(Al-Shok and Baey ,2009) study it was V. cholerae serotype Ogawa in addition to the smaller sample size in their study. Uppal B N et al reported in endemic area, the cholera usually affecting the 1-4 years old age group ,and falls thereafter , as titer of serum antibody rises and increasing immunity is acquired (Uppal ,1999; Myron,1999 and Bioro,1999 ). This study found that cholera infection would be considered as one of the causes of diarrhea in children whose aged less than five years old and the V. cholerae could be one of etiological agent of diarrhea particularly when the condition was associated with poverty and poor sanitation. These data indicate a need for urgent studies to determine the social and environmental factors associated with cholera epidemics in endemic areas in these ages groups to achieve effective control in the future.

Table -2 showed that the incidence of V. cholerae diarrhea in Babylon was (1.07 %) in relation to all diarrheal cases due to other causes. This study indicates that V. cholerae was one of the possible pathogens when children from a cholera-affected area develop diarrhea. Following the cholera outbreak in 2007, the Ministry of Health, with the support of WHO had established 950 surveillance sites in Iraq. These sites report every two weeks an acute watery diarrhea and cholera cases(HNSOT report,2008). This need raising the health precaution measures as high as possible to diagnose Vibrio's cases and to detect it's carrier state because prompt diagnosis of cholera is of key importance to initiate effective therapy and to institute proper epidemiological measures that limit its spread. As the problem in the control of cholera is the undetected carriers of the disease who are usually a hidden source of infection in the community(WHO, 2005) and humans are the reservoir of the disease (Huq A. et al,1986) , so continuation of vibrio cholerae preventive measures is highly recommended to prevent the carriers state that finally highly limit the transmission of this pathogen. On other hand, a cholera preparedness and response plan should be developed and the laboratory and testing capacity of the Ministry should be enhanced.

Table (3) showed that V. cholerae 01 had a frequency rate of (78.65 %) in comparison to non agglutinable Vibrio (21.35 %) from all detectable Vibrio species in Babylon Province. This is consistent with other study(Obuku,2001). The only detected V. cholerae was V. cholerae 01, biotype El Tor, serotype Inaba . This finding indicate that there may be re-emergence of Vibrio cholerae in the ongoing period because the isolated strain was of El Tor biotype which have a better survival in the environment and in the human host (Nesper, 2001), and more efficient host to host transmission of El Tor strains than of classical strains (Dougan, et al, 2002). This was consistent with Hasson S.O. et al who showed that the only detectable V. cholerae serotype in Babylon was serotype Inaba (Hasson, 2012). Other study done in Hamadan province in the west of the Islamic Republic of Iran in 2005, found that isolates have serotype Inaba (Keramat, 2008). This result may be explained as Iraq is at risk of epidemics spreading from neighboring countries because it lies on the routes of pilgrimage to holy shrines. This finding is in agreement with a study done in Pakistan, which showed that Inaba become the predominant serotype in 2005 outbreak (before, the Ogawa was the predominant serotype)( Jabeen,2008). However, this result may be not in agreement with a study done in Baghdad during 1999 outbreak which showed that Ogawa serotype is the predominant one(Al–abbassi A.M,2005). This may indicate that there is a change in V. cholera serotype epidemiology in the past decade.

Table (4) showed the autoagglutination pattern of V. cholerae which done for the detection of TCP (Toxin coregulated pili). It was found that 12.75% of V. cholerae isolates had mild autoagglutination while 21.81% have moderate autoagglutination and 65.43% have high autoagglutination. The appearance of low level of autoagglutination in this study may be due to a defect in pili biosynthesis pathway in agreement with Kaufman (Kaufman, and Taylor, 1994) and Nesper et al (Nesper,2001) who characterized and found that the galU and galE mutants gene and rough-LPS mutant comprising altered core oligosaccharide or it may be due to environmental signals including temperature, pH, osmolarity, and certain amino acids. It is reasonable to expect that the environmental conditions that exist in the GI tract (i.e., 37o temperature, low pH, high osmolarity, etc.) as opposed to conditions in the extra intestinal (aquatic) environment of the vibrios, that are necessary to induce formation of the virulence factors necessary to infect. However, there is conflicting experimental evidence in this regard, which leads to speculation of the ecological function of the toxin during human infection (Todar K.,2012). An understanding of this intricate signaling pathway is essential for the development of methods to treat and prevent this devastating disease (Finkelstein,1992). Also, several genes from the ancestral genome have functions in the normal bacterial physiology and have been found to have global effect in the control expression of the virulence genes. In addition to this finding, those patients with mild autoagglutination lose fluid to lesser extent than those with moderate or high autoagglutination, so this reflect the role of TCP as a major virulent factor in this bacteria which can be used as a target in the goals of vaccine preparation.