Laboratory Diagnosis of HIV-Opportunistic Infections - Laboratory Diagnosis of Viral Opportunistic Infections in HIV-Infected Patients

Guidelines on Standard Operating Procedures for Laboratory Diagnosis of HIV-Opportunistic Infections

Standard Operating Procedures for Laboratory Diagnosis of Viral Opportunistic Infections in HIV-Infected Patients

Introduction

Clinical Manifestations

Laboratory Diagnosis

Safety Considerations

Specimen Collection

Specimen Transport And Storage

This SOP is confined to simple techniques for diagnosis of herpes simplex virus (HSV), varicella-zoster virus (VZV) and cytomegalovirus (CMV) which are the common opportunistic viral infections in HIV-infected persons.

Clinical manifestations

Herpes simplex virus type 1 (HSV 1) and type 2 (HSV 2) infections cause primary and recurrent oral, genital and rectal ulceration and occasionally disseminated visceral and central nervous system disease.

VZV causes varicella in primary infection, and herpes zoster when reinfected later in lifetime. In HIV-infected persons, reactivation of VZV cause prolonged and severe manifestation of herpes zoster and occasionally becomes disseminated.

CMV infection is responsible for a greater proportion of opportunistic infections in HIV-infected persons with advanced immune deficiency. Retinitis is the most frequent clinical manifestation of CMV infection in patients with AIDS. Gastrointestinal disease, encephalitis, polyradiculitis, and pneumonia may also occur.

Laboratory diagnosis

Viral diagnosis is mostly based on clinical symptoms and signs. The most common technique is simple microscopic examination of scraped cells for diagnosis of HSV skin, oral, genital and anal lesions and VZV lesions (Tzanck test). Demonstration of multinucleated giant cells indicates infection by either HSV or VZV. The two viruses could be differentiated by immunological techniques i.e. immuno-fluorescence, immunoperoxidase and ELISA.

Detection of CMV can be done by conventional culture, PCR or the semiquantitative antigenemia assay. Demonstration of the virus in affected tissue confirms the diagnosis of CMV disease (e.g. bronchoalveolar lavage specimen, or biopsy and necropsy specimens). Detection of a high level of the virus in blood by a quantitative assay is predictive of CMV disease. Viral shedding in urine indicates reactivation and may be associated with CMV disease.

Serological diagnosis is useful in primary herpes virus infection, but not in reactivation and immunodeficiency states.

Viral culture and PCR are the techniques available in reference laboratories. Specimen collection, transportation and storage are described in this SOP.

The other viruses associated with HIV-infected persons are: Epstein Barr virus (EBV); Kaposi’s sarcoma-associated herpes virus (HHV 8); Other viruses, e.g. human papillomaviruses (HPV), molluscum contagiosum virus, hepatitis B virus (HBV), and hepatitis C virus (HCV). Investigation of these viruses is carried out in the reference and research laboratories.

Safety considerations

Specimen collection	Universal precautions and avoidance of contact with lesion or tissue with ungloved hands.
Specimen transport and storage	Sterile leak-proof container in a sealed plastic bag.
Specimen processing	Biosafety level II or III with good laboratory practice.

The above guidelines should be supplemented with recommendations from the local health hazard and risk assessment committee.

Specimen collection

Optimal time of specimen collection

At the time close to onset or as early as possible.

Before the start of antiherpetic drugs.

Correct specimen type and method of collection

Skin and mucosa lesions: Collect from newly formed vesicles. Using sterile No. 27 needle and tuberculin syringe, aspirate the vesicular fluid for culture. Open the lesion and scrape the cells at the rim of the base of the lesion. Smear on dry clean glass slides (2-3 slides)

Swab from the open lesion, immerse in viral transport media.

Cerebrospinal fluid: Collect 1-2 ml in sterile container.

Biopsy or necropsy tissues: Try to avoid contamination as much as possible. Collect in sterile container.

Blood: Collect venous blood 3-5 ml in EDTA tube.

Bronchoalveolar lavage: Collect 10 ml in sterile container.

Specimen transport and storage

Time between specimen collection and processing

All specimens should be fresh and sent to the laboratory as soon as possible.

Slides from skin or mucosal lesion smears should be air dried and sent in sealed plastic bags.

Vesicular fluid and tissue specimens should be kept in sterile containers and sealed plastic bags, kept refrigerated and transported in an ice-box.

Lesion swabs should be put in sterile tubes with appropriate amounts of viral transport media, kept in refrigerator and transported in an ice-box.

Blood specimen should be transported at room temperature.

Special considerations to minimize deterioration

Specimens should be processed as soon as possible especially for viral isolation.

For CMV antigenemia assay, specimens should be processed within six hours.

Specimen processing

Proper documentation upon receipt of specimen

Laboratory request form should indicate the specific test for HSV, VZV and CMV.

Record in laboratory file and give laboratory number.

Processing

Smears are fixed in absolute methanol and stained with Giemsa stain.

Smears are fixed in acetone and sent to a reference laboratory for staining with specific anti-HSV or anti-VZV as requested.

Specimens requested for PCR, viral culture and CMV antigenemia assay are referred to a reference laboratory.

Reporting procedure

Microscopy

Giemsa stain : Positive or Negative for multinucleated giant cell.Inadequate specimen if no epithelial cell is found.

Giemsa staining

To prepare Giemsa stock solution, dissolve 0.5 gm of Giemsa powder in 33 ml of glycerine by placing the mixture in a water bath (55o-60oC) for 90 minutes. When the powder is dissolved, add 33 ml of absolute methanol. The stock solution is stored at room temperature.

The scraping prepared onto clean glass slide is fixed in absolute methanol for ten minutes. The slides are then stained with Giemsa stain (freshly prepared by diluting the stock solution 1:20 in water) for two hours. The stained slides are decolourized in 95% ethanol for a few seconds and rinsed in tap water.

Reference

WHO(2000) Malaria. In Guidelines on Standard Operating Procedures for Microbiology (SEA/HLM/324) WHO Regional Office for South-East Asia, New Delhi, p. 119.