ABSTRACT

Neonatal sepsis is an invasive infection occurring in the first twenty eight (28) days of life.  It could be bacterial, viral, fungal or even toxin mediated (Okolo et al., 2015; Onyedibe et al., 2015). The mortality rate in neonatal sepsis may be as high as 50% for untreated infants (Okolo et al., 2015; Onyedibe et al., 2015). Infection is a major cause of fatality during the neonatal period. Neonates particularly those with very low birth weight (VLBW) usually require invasive therapies, such as catheterization, exchange blood transfusion, endotracheal intubation and these factors place them at high risk for fungal infection (Chitnis et al., 2012; Kauffman,2012). This will be a cross sectional study involving neonates admitted into the special care baby unit of the Jos University Teaching Hospital. Blood samples will be collected from neonates with septicaemia and aseptically inoculated into blood culture bottles. The samples will be transported immediately to the laboratory and put into the incubator. The blood culture bottles will be incubated for early identification of positive culture bottles which will be subcultured onto appropriate mycological media for 18 to 24 hours. Phenotypic identification will be carried out using both light and fluorescent microscopes. Antifungal susceptibility test, resistance gene testing as well as minimum inhibitory concentration will be determined following Clinical and Laboratory Standard Institute guidelines. Molecular typing as well as sequencing of amplified genes will be carried out using guided standard protocols. Phylogenetic analysis will be done following blasting of the genes using appropriate software. Early detection of neonatal fungal sepsis and appropriate treatment will reduce morbidity and mortality particularly in this part of the world where neonatal fungal infections are not well researched.

 

BUDGET:

DESCRIPTION OF ITEMS	Cost	INSTITUTION	OTHER	TOTAL
1.0 Personnel Cost / Allowances				NIL
2.0 Equipment/Consumables
Indian ink	2,000 x1			2,000
Lactophenol cotton blue	5,000 x2			10,000
Tween 80	20,000 x 1			20,000
Petri dish	12,000 x 5			60,000
Antifungal MIC discs	6,500 x 8			52,000
Mycological media:				155,000
Molecular Methods:     Primer sequences(Inqaba, S/africa) DNA extraction kits PCR/DNA sequencing	6,000 x10   250,000 x1 640,000			950,000
Sub-Total				N 1,249,000
ICTservices/ publications:	150,000			N  150,000
GRAND TOTAL				N 1,399,000

 

BUDGET JUSTIFICATION:

The personnel cost will not be covered by the research grant since I am on the university salary as well as the collaborators. Important consumables will be purchased for the research particularly for sample collection, culture and biochemical identification. 

A part of the research grant (10.7%) will be used for publications of research findings in reputable journals and STAMINA will be highly acknowledged.

However, the bulk of the budget will be used for the purchase of DNA extraction kits, primer sequence for targeted genes as well as for the molecular identification and phylogenetics.

1. PROJECT NARRATIVE:

            The awarding of these funds will assist in having base-line data for neonatal fungal sepsis, determination of newer minimum inhibitory concentrations and resistance genes that will set the stage for future studies. In addition, the results from this research will reduce the morbidity and mortality associated with fungal neonatal sepsis and set the stage for further epidemiologic studies.  

 

RESEARCH PLAN:

1. Specific aims

 

1. To determine the fungal agents causing neonatal sepsis in Jos University Teaching Hospital using molecular methods.
2. To determine antifungal susceptibility pattern of fungi isolated from the study population.
3. To Ascertain the Minimum Inhibitory Concentration of fungi isolated to aid the treatment of patients.
4. To determine resistance genes in the fungal isolates.
5. To carry out sequencing and Phylogenetic analysis of the fungal isolates.
6. To establish a research database for future references and studies.

BACKGROUND AND SIGNIFICANCE:

            Neonatal sepsis is an invasive infection occurring in the first twenty eight (28) days of life. Diagnosis is clinical, with extensive laboratory testing for confirmation and monitoring. Survival of neonates depend on advances in neonatal management. However, early (<72 hours of birth) and late (>72 hours of birth) onset of systemic infections due to bacterial and fungal agents, remain a devastating complication and an important cause of morbidity and mortality in these babies (Okolo et al.,2015; Onyedibe et al.,2015 ). Neonatal sepsis contributes up to 30-50% of all deaths during the neonatal periods in developing countries (Ballot et al.,2013; Onyedibe et al.,2015). The prevalence of neonatal sepsis in Jos, Nigeria is 34.4% (Onyedibe et al.,2015). An epidemiologic study of neonatal sepsis in Johannesburg hospital, South Africa showed prevalence of 6.3% for Candida albicans and 1.5% for Candida parapsilosis (Motara et al.,2005). There is paucity of data with regards to neonatal fungal sepsis in Nigeria. Intravenous antifungal agents continue to be the mainstay of therapy for systemic fungal infections (Chitnis et al., 2012; Kauffman,2012). 

            In March 2013, three different fungal organisms (Candida famata, Rhodotorulla species and Moesziomyces bullatus) were isolated from neonatal patients seen at the Jos University Teaching Hospital (Okolo et al.,2015). These are isolates that rarely cause human disease the world over. However, this discovery lead to the appropriate treatment of these neonates and ignited widespread research curiosity. Due to the lack of facilities for further identification in Jos, Nigeria, the research was carried out in collaboration with a research team in the mycology laboratories of the University of Messina, Italy; and C.B.S, Netherland for the molecular characterization, and antifungal susceptibility tests. The GenBank/EMBL/DDBJ accession number for the sequence in the study is KF926673 and already deposited in the gene bank in C.B.S, Netherland.

The isolation of Moesziomyces bullatus (M. bullatus) is a landmark achievement as it is the first time it has been identified as a cause of sepsis in humans in any part of the world. A closely related fungus Pseudozyma species, which belong to the same family as the isolated M. bullatus has been implicated as a cause of brain abscesses and cellulitis in humans (Hwang et al., 2010; Chen et al., 2011).

The laboratory processes involved in this study will provide database for future references, create protocol for fungal infection diagnosis and antifungal therapies, and provide fungal stock bank of isolates for future research.

PRELIMINARY DATA:

            The only available data is the finding of three fungal isolates obtained in the course of routine hospital investigations.

EXPERIMENTAL DESIGN AND METHODS:

             The research will be a prospective cross-sectional study involving neonates with septicaemia admitted into the special care baby unit (SCBU) of the Jos University Teaching Hospital which is a 600 bed tertiary hospital. The SCBU is a 30 bed unit for the management of neonates with special care needs.

Case definition:

All neonates with clinical diagnosis of neonatal sepsis whose parents consented to participate will be recruited based on the Integrated Management of Childhood Illnesses (IMCI) criteria: (see appendix).

Sample size:

Based on the local prevalence of 34.4%, using EPI-tools statistical software (http:/www.epitools.ausvet.com.au/content.php?page=samplesize) for sample size determination,  a minimum sample size of 345 was obtained using a confidence level of 95% and a desired precision of 0.05.

Inclusion criteria:

All neonates admitted into the SCBU on account of sepsis whose parents gave consent.

Exclusion criteria:

All neonates with sepsis who are already on antifungal therapy.

Sample Collection:

Blood samples will be collected from neonates with septicaemia and aseptically inoculated into blood culture bottles. The samples will be transported immediately to the laboratory and put into the incubator.

Laboratory studies:

The blood culture bottles will be incubated for early identification of positive culture bottles which will be sub-cultured onto appropriate mycological media for 18 to 24 hours. Phenotypic identification will be carried out using chromogenic media, biochemical reactions as well as light microscopy. Antifungal susceptibility test, minimum inhibitory concentration determination as well as phenotypic identification will be carried out in the JUTH medical microbiology laboratory following Clinical and Laboratory Standard Institute guidelines by the research team in JUTH. Bacteria isolated in the course of the research will be characterized, antibiotic susceptibility done and the result will be used for proper management of the patients.

Molecular methods:

Molecular typing as well as sequencing of amplified genes will be carried out using guided standard protocols. Phylogenetic analysis will be done following blasting of the genes using appropriate software. The molecular characterization will be carried out in collaboration with Dr Obishakin E.T at the molecular laboratory of the National Veterinary Research Institute Vom, which is situated at about 4 kilometers away from JUTH. The sequencing of resistance genes (ergII, kan MX3, kanMX4) will be carried out in Inqaba biotec laboratory (South Africa) from where the primer kits as well as the DNA extraction kits will be purchased.

Rare genes will be stored at the gene bank in  CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands, where as isolates will be preserved at -70^oC freezer as isolate banks for future research.       

Statistical analysis:

Statistical analysis will be carried out using epi-info 3.5.3 CDC Atlanta Georgia software. Continuous variables will be expressed as means while categorical variables will be expressed as absolute and relative frequencies. Chi-square test will be used to compare categorical variables.

2. REFERENCES:

 

1. Ojogba Mark Okolo, Anne D. van Diepeningen, Bose Toma, Nnaemeka Emmanuel Nnadi, Mebi Grace Ayanbimpe, Ikenna Kenneth Onyedibe, Mohammed Zaino Sabitu, Benle Edmund Banwat, Marizeth Groenewald, Fabio Scordino, Zanyul Daniel Egah, Giuseppe Criseo and Orazio Romeo (2015) First report of neonatal sepsis due to Moesziomyces bullatus in a preterm low-birth-weight infant. JMM Case Reports, 2(2): 1-4
2. Kenneth Ikenna Onyedibe, Fidelia Bode-Thomas, Tolulope Olumide Afolaranmi, Mark Ojogba Okolo, Edmund B. Banwat  and Daniel Zanyu Egah. (2015) Bacteriologic Profile, Antibiotic Regimen and Clinical Outcome of Neonatal Sepsis in a University Teaching Hospital in North Central Nigeria. BJMMR, 7(7): 567-579.
3. Chitins AS, Magill SS, Edwards JR, Chiller TM, Fridkin SK, Lessa FC. (2012) Trends in Candida central line-associated blood stream infections among NICUs, 1999-2009. Pediatrics, 130(1): 46-52.
4. Kauffman DA. (2012) “Getting to zero”: preventing invasive Candida infections and eliminating infection-related mortality and morbidity in extremely preterm infants. Early Hum Dev., 88(2)45-49.
5. Ballot DE, Bosman N, Nana T, Ramdin T, Cooper PA. (2013) Background changing patterns of neonatal fungal sepsis in a developing country. Journal of tropical paediatrics, 59(6): 460-464. doi 10.1093/tropej/fmt 053.
6. Motara F, Ballot DE, Perovic O. (2005) Epidemiology of neonatal sepsis at Johannesburg Hospital. South African Journal of Epidemiology and Infection;20(3):90-93.
7.  Hwang S, Kim J, Yoon S, Cha Y, Kim M, Yong D, Chang JH, Jeong SH, Uh Y, Lee K. 2010. First report of brain abscess associated with Pseudozyma species in a patient with astrocytoma. Korean J Lab Med. 30(3):284-8.
8. Chen B, Zhu LY, Xuan X, Wu LJ, Zhou TL, Zhang XQ, Li BX. 2011. Isolation of both Pseudozyma aphidis and Nocardia otitidiscaviarum from a mycetoma on the leg. Int J Dermatol. 50:714-9.
9. WHO handbook on Integrated Management of Childhood Illnesses (IMCI)   http:/www.who.int>topics>child>imci

 

3. Plan for Protection of Human Subjects:

A written informed consent will be obtained from the parents of the neonates prior to enrolment. All informations obtained will be kept confidential. The research involves collection of 1.5ml of blood from the neonates and will be of minimal risk and slight pain will be felt during collection. The pain will ease off within 10minutes of sample collection.  

 

4. Project Mentor: Prof. Ayanbimpe Grace Mebi , Department of Medical Microbiology, University of Jos,

 

4. Pending IRB Approval:

Ethical clearance for the study will be obtained from the Jos University Teaching Hospital Ethical Committee following a written application for approval to conduct the study.

 

APPENDIX  

IMCI criteria:

Fever (>37.5oC or <35.5oC)

Convulsions

Nasal flaring

Bulging fontanelle

Pus draining from ear

Skin pustules

Lethargy

Umbilical redness