Investigations

1st investigations to order

CD4 cell count

Test
Result
Test

The CD4 count is a highly sensitive and specific marker that determines the susceptibility to particular opportunistic infections.[77]​ Tuberculosis can occur throughout the course of HIV disease but the risk increases as the CD4 count decreases.[1][47]​​[78]Individuals with a CD4 count below 250 cells/microlitre living in or visiting areas in which Coccidioides species are endemic are at increased risk for coccidioidomycosis.[42]​ Pneumocystis jirovecii pneumonia and candidiasis are more likely when the CD4 count is below 200 cells/microlitre. Individuals with a CD4 count below 150 cells/microlitre who live in or have travelled to areas endemic for Histoplasma capsulatum are at increased risk for developing progressive disseminated histoplasmosis.[1]​ When the CD4 count is below 100 cells/microlitre, people are at a higher risk for toxoplasmosis.[1]​ Cytomegalovirus end-organ disease and Mycobacterium avium complex are more likely with CD4 count below 50 cells/microlitre.[1]​ More than three-quarters of cryptococcal infections associated with AIDS develop when the CD4 count falls below 50 cells/microlitre.[54]

Result

variable

sputum stain and culture

Test
Result
Test

In diagnosis of tuberculosis (TB) three sputum specimens should be obtained for acid-fast bacilli (AFB) smear and cultured.[82]

Examination of induced sputum is recommended as the initial screening test for Pneumocystis jirovecii pneumonia (PCP). Specimens should be evaluated with particular staining methods (e.g., Gomori-methenamine silver, Wright-Giemsa, Diff-Quick, or immunofluorescence). Since the negative predictive value of this test is relatively low, a negative test should prompt a referral for fibreoptic bronchoscopy with BAL, which significantly increases the diagnostic yield to >90%.[108] 

Sputum culture is a definitive test for coccidioidomycosis infection. Isolation of Coccidioides organisms from a patient’s sputum or other clinical specimen definitively establishes the diagnosis of coccidioidomycosis and should not be considered to represent colonisation. Organisms grow well within 5-7 days of incubation on most mycological and bacteriological media. When growth is noted, appropriate biocontainment measures are imperative, as cultures are highly infectious and laboratory personnel are at risk of infection.

Result

AFB stain and mycobacterial culture are positive in TB and MAC; staining is positive in PCP; positive in coccidioidomycosis infection

blood cultures

Test
Result
Test

Two blood cultures are usually sufficient for the detection of disseminated Mycobacterium avium complex (MAC). One blood culture identifies 91% of people with MAC bacteraemia, while a second blood culture increases the yield to 98%.[177] However, up to 6 weeks of culture may be required, which limits the clinical utility.

Three-quarters of people with HIV-associated cryptococcosis have positive blood cultures for Cryptococcus neoformans.[140]​ Blood cultures may be helpful if disseminated disease is suspected in the absence of meningitis.

Mycobacterial blood cultures may be positive in disseminated TB.[97]

Blood cultures may be particularly useful for diagnosing disseminated histoplasmosis, especially in severely immunosuppressed individuals. Culture is a definitive diagnostic method for Histoplasma capsulatum.[1]

Result

may be positive in MAC cryptococcus infection, disseminated TB, and disseminated histoplasmosis

adenosine deaminase

Test
Result
Test

Measuring adenosine deaminase in pleural fluid is an easy and inexpensive test that may help establish the diagnosis of pleural tuberculosis when significantly elevated.[92] Adenosine deaminase sensitivity was 94% and specificity 95% in people living with HIV, regardless of CD4 counts, when the cut-off value was 30 U/L.[93]

Result

elevated in TB pleuritis

Toxoplasma gondii serology

Test
Result
Test

Presence of anti-T gondii IgM antibodies does not necessarily indicate a recently acquired infection as they can remain elevated for more than 1 year. Recent T gondii infection is likely when serial specimens obtained at least 3 weeks apart and tested in parallel reveal at least a 4-fold increase in IgG titres.[178]​ A negative T gondii IgG makes toxoplasmic encephalitis significantly less likely but does not rule it out.[1]

Result

anti-T gondii IgM suggests recent infection and anti-T gondii IgG suggests past infection

Coccidioides serology

Test
Result
Test

Serological tests are most frequently used to diagnose primary pulmonary coccidioidomycosis and are highly specific for infection.[151] If serological testing is negative in suspected infection, repeat serological testing is recommended since it can take several weeks to develop an adequate antibody response.

Coccidioides serology should be performed in individuals with compatible clinical presentations if they have resided in or visited endemic regions.

Enzyme immunoassay (EIA) is widely available and detects specific IgM and IgG antibodies against Coccidioides. Positive EIA results should be confirmed with immunodiffusion and complement fixation.[1]

Result

positive IgM and/or IgG

cryptococcal polysaccharide antigen

Test
Result
Test

Very specific and highly sensitive (>90%). High serum titers (≥1:160) indicates a higher risk for CNS disease in asymptomatic individuals.[179] High initial cerebrospinal fluid (CSF) titres (≥1:1024) are a marker of poor prognosis and correspond to a high organism burden. CSF antigen titres typically fall with successful treatment.[160]

Result

from serum or CSF

Histoplasma capsulatum antigen

Test
Result
Test

Antigen detection tests for H capsulatum are the preferred diagnostic method for disseminated histoplasmosis. These assays demonstrate high sensitivity and specificity, particularly in disseminated cases. They allow for rapid diagnosis, reducing the time to treatment initiation.

Urine or blood samples are the preferred detection methods. Antigen testing may also be performed on cerebrospinal fluid and bronchoalveolar lavage fluid in cases involving the central nervous system or lungs.[1]

Result

Positive for H capsulatum

Histoplasma capsulatum culture

Test
Result
Test

Culture is a definitive diagnostic method for H capsulatum, but requires several weeks for results due to the slow growth of the organism. H capsulatum can be cultured from blood, bone marrow or respiratory secretions. Blood cultures may be particularly useful for diagnosing disseminated histoplasmosis, especially in severely immunosuppressed individuals.[1]

Result

Positive for H capsulatum

FBC

Test
Result
Test

Anaemia is common in people living with HIV with opportunistic infections. In disseminated Mycobacterium avium complex, the anaemia is an important negative predictor for survival, is usually profound, and is often accompanied by leukopenia and thrombocytopenia.[180][181]

Result

anaemia often accompanied by leukopenia and thrombocytopenia

LFT

Test
Result
Test

Alkaline phosphatase is typically elevated in people living with HIV with Mycobacterium avium complex or cytomegalovirus (CMV) hepatobiliary disease.[182] In CMV hepatitis, it is usually accompanied by elevation of aspartate aminotransferase and alanine aminotransferase.[183]

Result

elevated

LDH

Test
Result
Test

Often elevated in disseminated  Mycobacterium avium complex infection, and frequently elevated in Pneumocystis jirovecii pneumonia (PCP).

However, elevated LDH should be interpreted with caution, because it is also elevated in people with various other lung diseases, such as pulmonary tuberculosis and other bacterial pneumonias, as well as a variety of non-infectious conditions.[109][182]

Result

elevated (but non-specific)

ABG

Test
Result
Test

The arterial oxygen pressures (PaO₂) and P(alveolar-arterial [A-a])O₂ gradient correlate with prognosis. A P(A-a)O₂ of greater than 35 mmHg or a PaO₂ of less than 70 mmHg are associated with poorer outcome, and provide an indication for adjunctive corticosteroid therapy.[106]

Result

increased alveolar-arterial oxygen gradient, P(A-a)O₂; PaO₂ values vary widely depending on disease severity

CXR

Test
Result
Test

Should be performed in any person living with HIV with pulmonary symptoms.

Findings in people living with HIV with tuberculosis (TB) and a CD4 above 200 cells/microlitre: upper lobe infiltrates and cavitation; a CD4 below 200 cells/microlitre: mediastinal adenopathy is common and probably reflects an ineffective immune response.[2][154]

The prevalence of normal CXRs in individuals with confirmed Pneumocystis jirovecii pneumonia (PCP) is about 10%.[157] Various atypical radiographic manifestations have been reported, including nodular densities and cavitary lesions. Pneumothorax can occur, and its management is often difficult.[184] If the CXR appears normal but PCP is suspected, a high-resolution CT should be ordered.

Imaging studies are typically useful during the initial evaluation of coccidioidomycosis, but radiographic findings are non-specific.

CXR may be used in subsequent monitoring for improvement.[185]

Result

variable; TB: upper lobe infiltrates and cavitation or mediastinal adenopathy; PCP: bilateral diffuse infiltrates beginning in the perihilar regions; cytomegalovirus: interstitial infiltrate; histoplasmosis: diffuse bilateral reticulonodular infiltrates

head CT

Test
Result
Test

Should be ordered in all individuals with altered mental status.

In toxoplasmic encephalitis, lesions tend to involve the basal ganglia and hemispheric corticomedullary junction.[186]

In cryptococcal meningitis, CT findings may include single or multiple nodules (cryptococcomas), cerebral oedema, or hydrocephalus.[160]

Result

toxoplasma encephalitis: multiple, bilateral, hypodense, ring-enhancing lesions; cryptococcal disease: normal or may show meningeal enhancement; tuberculosis meningitis: tuberculomas, post-contrast basal enhancement, hydrocephalus, and infarcts

Investigations to consider

tuberculosis (TB) nucleic acid amplification

Test
Result
Test

Several rapid nucleic acid amplification tests (NAATs) are available for the diagnosis of TB, and some are also able to detect resistance to some TB drugs. Although NAATs were originally designed and approved for respiratory specimens, they may also be requested on specimens from other sites where involvement of TB is suspected (e.g., cerebrospinal fluid, lymph node aspirate, lymph node biopsy, pleural fluid, peritoneal fluid, pericardial fluid, synovial fluid, or urine).[83] In the US, use of NAATs for extrapulmonary specimens is not approved by the US Food and Drug Administration and therefore use in this setting is off-label.

Result

positive in TB infection

lateral flow urine lipoarabinomannan (LF-LAM) assay

Test
Result
Test

Lateral flow tests that detect lipoarabinomannan (LAM) antigen in urine are potential point-of-care tests for TB. One Cochrane review found the lateral flow urine lipoarabinomannan (LF-LAM) assay to have a sensitivity of 42% in diagnosing TB in HIV-positive individuals with TB symptoms, and 35% in HIV-positive individuals not assessed for TB symptoms.[90] The World Health Organization (WHO) recommends that LF-LAM can be used to assist in the diagnosis of active TB in HIV-positive individuals.[83] This approach is supported by another Cochrane review, which found reductions in mortality and an increase in treatment initiation with use of LF-LAM in inpatient and outpatient settings.[91]

Culture would still be required for drug susceptibility testing (DST).

Result

positive

bronchoalveolar lavage (BAL)

Test
Result
Test

Bronchoscopy may be required in individuals unable to produce sputum.

Extra caution should be exercised by the pulmonologist performing the BAL if tuberculosis is a consideration.[187][188][189]

Result

staining is positive in PCP; may be positive in disseminated cryptococcosis

oropharyngeal scrapings (KOH preparation) and culture

Test
Result
Test

Potassium hydroxide 10% (KOH) slide preparation can confirm the diagnosis of candidiasis. Pseudohyphae and budding yeast are characteristic findings.

Cultures alone are not diagnostic because colonisation is common and are usually not necessary unless the lesions fail to resolve on standard antifungal treatment.[190]

Result

presence of Candida species

cerebrospinal fluid (CSF) analysis

Test
Result
Test

Due to the risk of brain herniation if mass effect is present, caution should be exercised when considering lumbar puncture.[178]

Toxoplasmosis infection: Giemsa staining can show tachyzoites.

Cryptococcal meningitis: India ink examination is positive in 70% to 90% of people living with HIV. CSF culture requires 48-72 hours. The opening pressure in the CSF is elevated in up to 75% of individuals; CSF pressures above 250 mm of H₂O may require large-volume CSF drainage.[160][172]​​​ CSF cryptococcal antigen is also important to include, as it is a very sensitive and specific test and can be rapidly performed.

Result

toxoplasma encephalitis: mild pleocytosis of mononuclear predominance and elevated protein; cryptococcal meningitis: WBC elevated with lymphocytosis, elevated protein, and low glucose

polymerase chain reaction (PCR): cerebrospinal fluid (CSF), bronchoalveolar lavage (BAL) fluid, and vitreous and aqueous humor specimen for Toxoplasma gondii

Test
Result
Test

Can be useful in the diagnosis of toxoplasmic encephalitis. PCR in CSF has a sensitivity that varies from 12% to 70% and a specificity of approximately 100% in individuals with toxoplasmic encephalitis. A positive PCR in brain tissue does not necessarily indicate active infection because tissue cysts persist in the brain long after acute infection.[191]

Result

positive

bone marrow aspirate and culture

Test
Result
Test

May be helpful in the diagnosis of disseminated Mycobacterium avium complex and tuberculosis, although acid-fast bacilli (AFB) blood cultures are the test of choice. Diagnostic sensitivity of bone marrow cultures may be no greater than that of blood cultures in these individuals, but the histopathological examination of bone marrow specimens results in the relatively rapid identification of infections in some individuals who are culture negative.[192] In early disease, the sensitivity of bone marrow culture may actually be higher than that of AFB blood culture.

Result

granulomas; organism seen on acid-fast stain

lymph node aspirate or biopsy

Test
Result
Test

Lymph node fine-needle aspiration or biopsy allows a specific diagnosis in 100% of individuals with Mycobacterium avium complex focal lymphadenitis.[193]

Result

granulomas; organism seen on acid-fast stain

tissue biopsy

Test
Result
Test

Should be considered when other less invasive tests have been non-diagnostic and the pulmonary process is progressive.

In individuals with suspected tuberculosis (TB), trans-bronchial biopsy or biopsy of an extrapulmonary site (e.g., bone marrow, vertebral column) may be required. On pathological examination tuberculous granulomas are detected in 60% to 100% of cases, depending on the person's immune status and the site of sampling.[96]

The specimen obtained should also be examined for acid-fast bacilli (AFB) and cultured for mycobacteria.

Individuals with clinical illness suggestive of tissue-invasive cytomegalovirus (CMV) disease should have tissue specimen (liver, intestinal mucosa, lung) obtained by biopsy for the detection of CMV.[194]

Positive histopathology gives a definitive diagnosis of coccidioidomycosis.

Periodic acid-Schiff or Gomori methenamine silver staining may be used to identify Histoplasma capsulatum. Histopathology is particularly useful for rapid presumptive diagnosis in acute clinical scenarios of histoplasmosis.[27]

Result

TB: granulomas, visualisation of AFB; CMV: demonstration of CMV-specific cytoplasmic and intra-nuclear inclusions in liver, intestinal mucosa, or lung specimens; coccidioidomycosis: identification of spherules on microscopy; histoplasmosis: H capsulatum appear as relatively small (2-4 microns), ovoid, intracellular yeast

brain biopsy

Test
Result
Test

Can provide a definitive diagnosis of toxoplasmic encephalitis.

Excisional brain biopsy findings range from granulomatous reaction with gliosis and microglial nodules to necrotising encephalitis. The presence of tachyzoites or cysts surrounded by inflammation is considered diagnostic.[159]

Result

toxoplasmic encephalitis: presence of tachyzoites or cysts surrounded by inflammation

abdominal CT

Test
Result
Test

Consider in individuals with abdominal pain or if disseminated Mycobacterium avium complex (MAC) is suspected.

The CT scan in abdominal tuberculosis (TB) usually shows ascites and omental thickening.[154][195] Hepatomegaly, splenomegaly, and small wall thickening may also be noted.[105]

Result

abdominal TB: visceral lesions and intra-abdominal lymphadenopathy with necrosis; disseminated MAC: enlarged intra-abdominal mesenteric and/or retro-peritoneal lymph nodes

high-resolution CT (HRCT) of the chest

Test
Result
Test

When chest radiographic findings are normal, in someone with possible Pneumocystis jirovecii pneumonia (PCP), HRCT, which is more sensitive than chest x-ray, may reveal more faint ground-glass opacities or other subtle lesions. The sensitivity, specificity, positive predictive value, and negative predictive value of HRCT for the diagnosis of PCP are 100%, 83.3%, 90.5%, and 100%, respectively. HRCT is a reliable method for differentiating PCP from other infectious processes in HIV-positive individuals and a good method to rule out PCP.[115][184]​​[196]

Findings include bilateral ground-glass opacities, patchy bilateral consolidation, and multiple nodules or mass-like areas of consolidation.[197]

Ground-glass opacities can be homogeneous (24% of episodes), spare the lung periphery (41%), or display a mosaic pattern (29%). Other findings can include: reticulation, tree-in-bud appearance, consolidation, or cystic lesions.[197]

Result

PCP: ground-glass opacities; cytomegalovirus: diffuse interstitial and parenchymal changes

polymerase chain reaction (PCR) for Pneumocystis jirovecii pneumonia (PCP)

Test
Result
Test

PCR may be used to aid in the diagnosis of PCP. While a positive test may reflect colonisation rather than active disease, a negative test from a lower respiratory tract specimen can be used to confidently exclude PCP.[1][112]

Result

Negative test excludes PCP

beta-D-glucan

Test
Result
Test

Beta-D-glucan, a cell wall component of most fungi, may be useful for non-invasive diagnosis of Pneumocystis jirovecii pneumonia (PCP). Although elevated beta-D-glucan is not specific to PCP, it should raise suspicion if raised in a person with risk factors and a compatible clinical presentation.[112]

Result

Elevated (but non-specific for PCP)

brain MRI

Test
Result
Test

More sensitive than CT scan for toxoplasmic encephalitis and the preferred imaging technique, especially in individuals without focal neurological abnormalities. Individuals with only one lesion or no lesions apparent on CT scan should undergo MRI to determine whether more than one lesion is present.[121]

MRI scan is more sensitive for detecting multiple enhancing nodules within the brain parenchyma, meninges, basal ganglia, and midbrain.[160]

Result

toxoplasmic encephalitis: multiple or solitary focal brain lesions; cryptococcal disease: multiple enhancing nodules

thallium single photon emission tomography and PET

Test
Result
Test

Useful for distinction between central nervous system (CNS) lymphoma and infectious processes in people living with HIV with focal brain lesions.[123]

Result

increased uptake is an indicator of malignancy (CNS lymphoma)

upper gastrointestinal endoscopy and colonoscopy

Test
Result
Test

On biopsy, cytomegalovirus (CMV) intra-nuclear inclusions are identified in the densely inflamed granulation tissue of the ulcer base.[198]

In oesophageal candidiasis, pathological findings include: desquamated superficial hyperplastic hyper-keratotic squamous epithelium and inflammatory cells, with infiltration by fungal elements.[199]

Result

CMV colitis: lesions vary from punctate and superficial erosions to deep ulcerations, with granular and friable intervening mucosa

coccidioidal antigen testing

Test
Result
Test

Coccidioidal antigen testing can be performed on serum, urine, and cerebrospinal fluid.

Result

positive for coccidioidal antigen

polymerase chain reaction (PCR) for Coccidioides

Test
Result
Test

Real-time PCR can be used for detection of Coccidioides although availability is generally limited.

Result

positive for coccidioidal DNA

polymerase chain reaction (PCR) for Histoplasma

Test
Result
Test

PCR-based molecular diagnostic methods demonstrate high sensitivity and specificity for detecting Histoplasma DNA. These methods can serve as adjunctive tests, particularly when conventional diagnostics yield inconclusive results.

However, availability and standardisation of molecular assays remain limited in clinical practice.

Result

Positive for Histoplasma DNA

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